Novel polypeptides and coagulation therapy

ABSTRACT

Novel polypeptides (NPs) are provided which are capable of protein C activation without significant fibrinogen clotting activity, and vice versa. NPs having enhanced protein C activating properties in relation to fibrinogen clotting are useful in particular as anticoagulants and in screening for substances that agonize or antagonize this property and in diagnostic procedures to determine the status of patients&#39; activated protein C-mediated anticoagulant pathway. Procoagulant NPs are useful to promote clotting in the course of therapy of solid tumors, as an impregnate for bandages, or in diagnostic assays. The NPs are produced in recombinant cell culture or by in vitro methods.

[0001] This is a continuation of U.S. Ser. No. 09/504,735, filed Feb.16, 2000, now pending, which is a divisional application of U.S. Pat.No. 6,110,721, which is a continuation-in-part of U.S. Ser. No.08/258,038, filed Jun. 10, 1994, abandoned, which in turn is acontinuation-in-part of U.S. Ser. No. 08/152,657, filed Nov. 12, 1993,abandoned.

BACKGROUND OF THE INVENTION

[0002] Thrombin, a key enzyme in hemostasis, has both procoagulant andanticoagulant properties, based on its different substratespecificities. Thrombin is secreted from the liver as an inactivezymogen, prothrombin, that is activated by coagulation factors Va and Xato yield mature α-thrombin. This process can be mimicked in vitro by theproteolytic cleavage of prothrombin with various snake venoms such asEchis carinatus venom.

[0003] Thrombin acts as a procoagulant by the proteolytic cleavage offibrinogen, ultimately resulting in the formation of an insoluble fibrinclot, the activation of the clotting cofactors factor V and Factor VIIIto FVa and FVIIIa, the cleavage of Factor XI to activated Factor XIa(leading to further activation of Factors IX and X and perpetuation ofclotting) and the cleavage of the platelet thrombin receptor, resultingin platelet activation. On the other hand, when thrombin binds tothrombomodulin (TM), an integral membrane protein on vascularendothelial cells, thrombin undergoes a conformational change such thatthrombin loses its procoagulant activity and instead acquires theability to convert a plasma protein called protein C (PC) to activatedprotein C (aPC). aPC, a serine protease, acts as a potent anticoagulantby inactivating activated FV (FVa) and FVIII (FVIIIa), two essentialcofactors in the clotting cascade. aPC also inactivates plasminogenactivator inhibitor-1 (PAI-1), the major physiologic inhibitor of tPA(tissue plasminogen activator), thus potentiating normal fibrinolysis.This mechanism may serve to ensure that blood coagulation remainslocalized at the site of injury. Infants completely deficient in PC areessentially incompatible with life, with a fatal thrombotic disordercalled neonatal purpura fulminans; some patients with a partialdeficiency of PC have recurrent thrombosis. In addition, many recentanimal models utilizing aPC infusion have shown that exogenous aPC is ananti-thrombotic and anti-inflammatory molecule.

[0004] Human thrombin is generated from a precursor polypeptide,prothrombin, of approximately 579 mature amino acids (subject topotential allelic variation or N-terminal microheterogeneity) plus apresequence of about 43 residues (Degen et al., “Biochemistry” 22:2087[1993]). The presequence is proteolytically removed by the cell duringthe process of expression and secretion of prothrombin. Prothrombin is azymogen, or inactive protease, that is activated by proteolyticcleavage. At least three basic sites are subject to cleavage. In vivo,prothrombin is cleaved between residues R271 and T272 (Degen et al.residue numbers) by Factor Xa in the presence of Factor Va, phospholipidand calcium ions to yield prethrombin 2 and Fragment 1.2. Prothrombinalso is proteolytically cleaved by the same system between R320 and I321to yield meizothrombin, which in turn is cleaved autolytically betweenR155 and S156 to produce Fragment 1 (1-155) and meizothrombin des 1 (adisulfide linked dipeptide extending from residue 156 to the carboxyterminus of prothrombin, cleaved at R323). Finally, thrombin isgenerated from prethrombin 2 by proteolytic cleavage between R320 and1321, or from meizothrombin des 1 by proteolytic cleavage between R271and T272. Thrombin itself then autolyzes cleavage between T284 and T285to generate the mature A-chain N-terminus. For the purposes herein, themature N-terminal residue of the thrombin A chain (Degen T285) isdesignated “T1a” and is then numbered consecutively to the arginineresidue at R36a. The B chain is numbered from its N-terminal residue I1(Degen I321)through E259. The two thrombin peptides are covalentlybonded by a disulfide linkage between C9a and C119.

[0005] Two distinct numbering systems are in use for thrombin, inaddition to the DNA-based system of Degen et al. One is based onalignment with chymotrypsinogen (Bode et al., “EMBOJ” 8:3467 (1989). Asecond is favored by Sadler and coworkers at the University ofWashington. The Sadler numbering scheme is used in this specification.Under this protocol, the B chain of thrombin commences with I1 andextends to E259, while the A chain is designated with “a” postscripts asnoted above, as in T1a to R36a. This thrombin is termed “referencesequence thrombin,” and its entire sequence is shown in FIG. 1. Forexample, Wu et al., (“PNAS USA” 88:6775, (1991)) disclose severalthrombin mutants numbered in accordance with the Sadler scheme. The Wuet al. mutants and the corresponding chymotrypsinogen and Degen et al.residue numbers, respectively, are sequentially shown as follows: H43(57,363), K52 (60f, 372), N53 (60g, 373), R62 (67, 382), R68 (73, 388),R70 (75, 390), D99 (102, 419) and S205 (195, 525).

[0006] It is known in the literature that the thrombin binding sites forfibrinogen and protein C activation are overlapping but not identical.This is based on a small number of thrombin mutants (Wu et al., op cit).Wu et al. reported that a polypeptide having the sequence of thrombinbut with glutamic acid substituted at position 52 (K52E) wasapproximately 2.5 fold more active in producing activated PC thanwild-type thrombin and possessed only about 17% of the normal fibrinogenclotting activity of wild-type thrombin. Conversely, a polypeptidehaving the sequence of thrombin but with glutamic acid substituted atposition 70 (R70E) reportedly had the fibrinogen clotting activity ofwild-type thrombin but only approximately 7% of the PC activatingcapability of wild-type thrombin. According to Wu et al., the R68Eprotein essentially lost both functions.

[0007] For other polypeptides having sequence homology to thrombin, seeLe Bonniec et al., “JBC” 268(25):19055 (1993); Le Bonniec et al., “JBC”266(21):13796 (1991); Le Bonniec et al., “PNAS USA” 88:7371 (1991);Sheehan et al., “JBC” 268(5):3639 (1993); Horrevoet et al., “JBC”268(2):779 (1993); Suzuki et al., “JBC” 266(28):18498 (1991); Sheehan etal., “Thrombosis and Haemostasis” 69:Abstract 1784 (1993); Gan et al.,“Thrombosis and Haemostasis” 69:Abstract 1783 (1993); Gan et al.,“Thrombosis and Haemostasis” 69:Abstract 1787 (1993), and Naray-Szabo etal., “Theochem” 59:401 (1989).

[0008] The pivotal role of thrombin in blood clotting has made thisprotein a target in the development of agents for the treatment ofthrombosis. Most efforts have focused on the direct inhibition of thethrombin proteolytic activity, and in fact numerous inhibitors of theprocoagulant activities of thrombin have an anticoagulant effect (Hirsh,1991; Hirsh, 1991a). However, the potency of these inhibitors may belimited by the concomitant inhibition of the anticoagulant activity ofthrombin. Conversely, anticoagulant effect has been achieved byaugmenting or stimulating the thrombin anticoagulant pathway, i.e., byadministration of soluble TM (Gomi, et al., “Blood” 75:1396-1399, 1990;and Light, D., WO 93/15755) or activated protein C (“aPC”) Dreyfus etal., 1991; Gruber et al., 1990; Gruber et al., 1991; Taylor et al.,1987). This strategy does not block ongoing coagulation resulting frompreviously activated thrombin.

[0009] It is an object of this invention to prepare novel polypeptideswhich possess enhanced physiochemical or biological activities.

[0010] A further object of this invention is to prepare novelpolypeptides in which the procoagulant and anticoagulant activities ofthrombin have been substantially segregated, and which also optionallyresist heparin-mediated antithrombin III (AT-III) inhibition.

[0011] Another object is to obtain such polypeptides that can beexpressed in elevated yields in recombinant cell culture.

[0012] An additional object of this invention is to provide novelpolypeptides that are substrate specific for protein C activation orfibrinogen, but do not substantially proteolyze polypeptides thatnormally are not thrombin substrates.

[0013] Another object of this invention is to provide novelcovalently-modified polypeptides that are useful in screening forsubstances that are agonists or antagonists of thrombin's procoagulantor anticoagulant activities.

[0014] A further object is to provide novel polypeptides that activateprotein C but are substantially incapable of, or have reducedproteolytic activity against any one or more of fibrinogen, the thrombinplatelet receptor, and/or Factors XIII, V, XI or VIII.

[0015] A still further object is to obtain novel polypeptides useful inpurifying thrombin-interactive polypeptides such as TM or aPC from cellculture or native sources.

[0016] Another object is to identify novel polypeptides retaining atleast a substantial degree of the desired proteolytic activity ofthrombin, including the kcat and Km of thrombin for the desiredsubstrate.

[0017] In a further object, novel polypeptides are provided that exhibitenhanced PAI-1 inactivating activity as compared to wild-type thrombin.

[0018] A further object is to provide a method for identifyingdeficiencies in thrombomodulin function in patients with clottingdisorders.

[0019] In other objects, novel polypeptides are provided for thetreatment of thrombosis, in particular thrombosis associated with septicshock, for the therapy of solid tumors and for the preparation ofimproved dressings for wounds, or for therapies and diagnostic utilitiesthat rely upon a property of thrombin.

[0020] Another object is to identify novel analogues of thrombin thathave an enhanced or reduced ability to stimulate cell proliferation(Ben-Sharit et al., “PNAS USA” 83:976-980 (1986)).

[0021] These and other objects of the invention will be apparent fromconsideration of this specification as a whole.

SUMMARY OF THE INVENTION

[0022] This invention is concerned with novel polypeptides (hereafter“NPs”) in which the properties of thrombin are segregated, i.e., wherethe polypeptides fail to possess to a substantial degree one or moreundesired properties of thrombin yet still retain one or more desirableproperty of thrombin. In addition, this invention is concerned with NPsin which targeted thrombin residues have been mutagenized.

[0023] Specifically excluded from the scope of the NPs are known aminoacid sequence variants of thrombin, specifically thrombin R70E, thrombinR68E, thrombin K154A, thrombin K252E, thrombin K174E, thrombin R180E,thrombin D99A, thrombin D99N, thrombin E202Q, thrombin E25K, thrombinR245E, thrombin S205A, R197E, D199E, thrombin N151D, K154E, thrombindesP48,P49,W50, thrombin desE146,T147,W148, thrombin desT147-S158, thethrombins of WO 93/13208 in which at least one amino acid residue withinthe thrombin activation site has been mutated, and thrombin in whichloop F19-E25 is replaced by the equivalent loop from tissue plasminogenactivator. However, thrombin K52E is only excluded to the extent thatits manufacture is enabled by the prior art. Activation site variantthrombins are excluded only to the extent the term “activation site” isdefined and disclosed in WO 93/13208. Also excluded from the scope ofthe novel polypeptides of this invention are the fusions of such knownthrombins with a nonthrombin polypeptide or a preprothrombin orprothrombin polypeptide. Not excluded from the scope of NPs, however,are NPs representing known thrombins in which additional amino acidsubstitutions, insertions or deletions have been made.

[0024] Also excluded from the scope of the novel polypeptides arenaturally-occurring thrombins, whether from humans or animals (includingnaturally-occurring alleles), which have not been isolated or purifiedfrom blood or other body tissue, i.e., which are products of nature. Itwill be understood, however, that the NPs of this invention includeallelic variations from the thrombin reference sequence in addition tothe contemplated mutations herein.

[0025] In certain embodiments of this invention, we provide novel,proteolytically active polypeptides which have a ratio of protein Cactivation to fibrinogen clotting that differs from wild-type thrombin.In particular, we provide two general classes of novel polypeptides. Inthe first class, called “Protein C Activators”, or “PCA”, we providenovel polypeptides that possess a ratio of anticoagulant activity toprocoagulant activity of great than about 2. PCA polypeptides are ableto activate protein C but are substantially unable to cleave fibrinogen.Surprisingly, our experimental studies in animals demonstrate that evenresidual levels of procoagulant activity in PCA are well-tolerated anddo not result in any evidence of disseminated intravascular coagulationor other clinically adverse procoagulant responses. Moreover, we haveunexpectedly been able to identify PCA that are devoid of any detectableprocoagulant activity in our assay but still are capable of substantialProtein C activation. Thus, an embodiment of this invention comprisesadministering to a subject in need of anticoagulant therapy atherapeutically effective dose of an PCA.

[0026] In an extension of the foregoing embodiment, an PCA is preparedand administered whose anticoagulant activity is substantially resistantto inhibition by a predetermined thrombin inhibitor, for example heparinand AT-III. In one embodiment the PCA is administered in vivo inconjunction with heparin. Heparin inhibits the procoagulant activity ofendogenous thrombin without affecting the anticoagulant activity of thePCA, thereby resulting in a potent anticoagulant effect. This embodimentof the invention has the additional advantage in that the administeredPCA is expected to be resistant to AT-III-heparin clearance in vivo andthus will exhibit a longer biological half-life.

[0027] In other embodiments PCAs are provided that have reducedproteolytic activity towards the platelet thrombin receptor and thus donot activate platelets at therapeutic doses. PCAs are provided that havean EC50 for the stimulation of platelet aggregation of greater than 10nM, ordinarily greater than 20 nM. EC50s greater than that of wild-typethrombin reduce or eliminate detectable platelet aggregation by the PCAwhen the PCA is used at doses capable of activating Protein C.

[0028] A second group of novel polypeptides of this invention are termed“Fibrinogen Clotting Proteins” or “FCP”. These polypeptides possess aratio of anticoagulant activity to procoagulant activity of less than05. These NPs contain mutations in the protein C activating domains ofthrombin which reduce the anticoagulant activity of the resultingpolypeptide to less than about half that of wild-type thrombin, butretain the ability to clot fibrinogen. These polypeptides are useful,for example, in diagnostics, preparative methods and hemostasticsurgical articles. A further embodiment of this invention comprisesadministering to a subject in need of procoagulant therapy atherapeutically effective dose of an FCP.

[0029] To the extent that any enabling disclosure of an FCP or PCA hasappeared in the prior art literature, such prior art polypeptides areuseful in the foregoing therapeutic methods of this invention.

[0030] In one embodiment we have provided PCA polypeptides wherein anamino acid residue of a thrombin polypeptide has been substituted,deleted or a residue inserted adjacent to one or more of thrombinresidues W50, K52, D58, K65, H66, Y71, N74, K106, K107, S176, T177,W227, D193, K196, E202, E229, R233, D232, D234, K236, Y237 or F239.

[0031] In another embodiment we have provided FCP polypeptides whereinan amino acid residue of a thrombin polypeptide has been substituted,deleted or a residue inserted adjacent to one or more of the thrombinresidues K21, Q24, R70, R98 or K77.

[0032] Another embodiment of this invention facilitates the specificdiagnosis of various idiopathic thrombotic disorders. This embodiment isa method comprising contacting a subject's blood and vascular tissuewith a diagnostically effective dose of an PCA, and thereafter measuringa hemostatic parameter of the subject's blood to determine whether adefect exists in the subject's aPC pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIGS. 1A-1C depicts the nucleotide sequence for DNA encodingreference sequence thrombin (also referred to as wild-type thrombin),its complementary sequence, and the deduced amino acid sequence ofreference sequence thrombin.

[0034]FIGS. 2A and 2B compare the activities in rabbits of recombinantwild-type human thrombin (FIG. 2A) and the K52A PCA (FIG. 2B). Thisstudy shows that the novel polypeptide, in comparison to wild-type humanthrombin, produces no significant reduction in circulating fibrinogenbut is able to induce anticoagulation, as measured by the activated PTTassay.

[0035]FIG. 3 shows that the K52A PCA has a significant half-life invivo, conferring anticoagulant activity for up to about 150 minutesafter administration.

[0036]FIG. 4 compares the anticoagulant activities of two doses of anovel polypeptide of this invention (PCA-2, E229A PCA) in cynomolgusmonkeys.

[0037]FIG. 5 compares the anticoagulant activities of two PCAs of thisinvention (K52A and E229A) in cynomolgus monkeys.

[0038]FIG. 6 demonstrates the continuing thrombomodulin dependence oftwo NPs of this invention, K52A and E229A.

[0039]FIG. 7 illustrates the substantially reduced potency for plateletactivation of two of the NPs of this invention as compared to wild-typethrombin.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The novel polypeptides of this invention have a polypeptidesequence that is at least about 80% homologous by amino acid sequence(ordinarily at least about 90%, and preferably at least about 95%) withreference sequence thrombin, but have a significant qualitative orquantitative property not possessed by reference sequence thrombin, asdescribed elsewhere herein.

[0041] “Homology” is defined as the percentage of residues in acandidate amino acid sequence that are identical with the residues inthe reference sequence thrombin after aligning the two sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One computer program which may be used or adapted for purposes ofdetermining whether a candidate sequence falls within this definition is“Align 2“, authored by Genentech, Inc., which was filed with userdocumentation in the United States Copyright Office, Washington, D.C.20559, on Dec. 10, 1991.

[0042] In calculating amino acid sequence homology the candidate andreference sequences are aligned in the fashion that produces the maximumnumber of aligned residues, with insertions and deletions of residuesrepresented by gaps in the aligned sequences. For example, a 120 residuepolypeptide containing a 100 residue thrombin reference sequencefragment fused at its N-terminus to a heterologous 20 residue, bacterialsignal sequence, but with a single substitution in the thrombinfragment, is calculated to be 99% homologous to the thrombin referencesequence since the sequence of the fragment corresponds exactly to themaximally aligned thrombin reference sequence except for a singleresidue substitution and the 20 residue N-terminal fusion. Thus, if thealignment-maximizing comparison of the candidate and reference sequencesreveals an insertion (or deletion) of one or more amino acid residues,then these residues are ignored for the purposes of making the homologycalculation.

[0043] In another example, the designation “E202A NP” as used hereinmeans that the polypeptide so designated includes alanine substitutionat thrombin B-chain site 202 including any of the following: maturehuman thrombin B chain (free of the A chain), human prothrombincontaining both A and B chain, a fusion of a bacterial polypeptide withthe human B chain thrombin, or a site 202-containing fragment of thehuman thrombin B chain, so long as each of these derivatives retains thecapability of activating protein C or cleaving fibrinogen to produce aclot, or can be processed to do so. Fragments of thrombin A or B chains,in particular of the B chain are included as well, again provided thatthe polypeptide in its entirety at least is capable of activatingprotein C or cleaving fibrinogen to produce a clot. Fragments ofthrombin range from about 10, 20, 30, 50, 100 or more residues.Generally, NPs that contain more than one substitution in the thrombinsequence also will include the intervening thrombin sequence.

[0044] Analysis of homology is based on any one or more of the sequenceimputed from the nucleic acid used to express the NP, the sequence ofthe product as first produced in vitro, or the sequence after anypost-translational modification. Thus, if the reference and candidatesequences are identical when expressed, but a glutamine residue is laterdeaminated to glutamic acid, the first candidate is 100% homologous, butthe deaminated sequence is not.

[0045] For the purposes herein “procoagulant activity” is defined as theactivity determined by the fibrinogen clotting assay of Example 2.3,corrected to normalize the concentration of NP and correspondingwild-type thrombin.

[0046] For the purposes herein “anticoagulant activity” is defined asthe activity determined by the protein C activation assay of Example2.4, corrected to normalize the concentration of NP and correspondingwild-type thrombin.

[0047] The concentration of NP and thrombin is determined by anysuitable assay. Table la below reports results in which theconcentrations of NP and thrombin proteins are inferred from amidolyticactivity. However, an immunoassay also is satisfactory for this purpose.For example, immobilized PPAK can be used to capture the NP andthrombin, and the bound proteins detected by labeled anti-thrombinantibody. If the NP or thrombin is substantially homogeneous then grossprotein assays such as the well-known Lowry method can be used todetermine their concentration in the test sample.

[0048] The “corresponding” wild-type thrombin means a protein that ismade by essentially the same method as the analyte NP was made and hasthe same sequence as the NP except that mutations found in the NP arereverted back to the sequence found in reference sequence thrombin.

[0049] In some embodiments novel polypeptides possess (a) at least oneimmune epitope that is capable of substantial cross-reaction with anantibody raised against reference sequence thrombin, (b) two chainshomologous to the A and B chains of the reference sequence thrombin thatare bonded by a single disulfide linkage, (c) the thrombin active siteresidues S205, H43 and D99, (d) a kcat for fibrinogen or protein C, asthe case may be, which is at least about 20% of, preferably at leastabout 75% of and most preferably greater than equal to that of thereference sequence, (e) a Km for fibrinogen or protein C, as the casemay be, which is at most about 150%, preferably at most about 100% andmost preferably at most about 50%, and preferably at least about 75%, ofthat of the reference sequence, (f) proteolytic activity as measured byS-2238 hydrolysis that is greater than about 50% of the referencesequence thrombin, preferably greater than about 75% and most preferablygreater than about 90% of the reference sequence thrombin, and/or (g)which essentially has no broader substrate specificity than referencesequence thrombin, e.g., which is able to cleave human proteins otherthan the native thrombin substrates in vivo at a rate of no more thanabout 150%, and preferably no more than about 120% of the referencesequence.

[0050] The PCA of this invention typically possess (a) procoagulantactivity that is equal to or less than about 50, 25, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1% of reference sequence thrombin, (b) a ratio ofanticoagulant activity to procoagulant activity that is greater thanabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25 or 50 when compared withreference sequence thrombin, and (c) anticoagulant activity that isequal to or greater than about 5, 10, 15, 25, 50, 75 or 100% of theanticoagulant activity of reference sequence thrombin.

[0051] The FCP of this invention typically possess (a) anticoagulantactivity that is equal to or less than about 50, 25, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1% of reference sequence thrombin, (b) a ratio ofanticoagulant activity to procoagulant activity that is less than about0.5, 0.25, 0.15, 0.10, 0.09 or 0.08, when compared with referencesequence thrombin, and (c) procoagulant activity that is equal to orgreater than about 5, 10, 15, 25, 50, 75 or 100% of the procoagulantactivity of reference sequence thrombin.

[0052] The novel polypeptides of this invention comprise substitutionsfor, deletions of, or insertions of any amino acid residue adjacent to,any of the reference sequence amino acid residue sites shown in Table 1or 1b below. Table 1 depicts the results of a study in which thrombinresidues have been substituted with alanine. Substitutional NPs arethose in which at least one amino acid residue in the reference sequencehas been removed and a different amino acid inserted in its place at thesame position. The substitutions may be single, where only one aminoacid in the molecule has been substituted, or they may be multiple,where two or more amino acids have been substituted at 2 or morethrombin sites. The Table 1 columns, from left to right, depict our codenumber, the residues substituted, two columns of S-2238 hydrolysis data(a measure of the proteolytic activity of thrombin and an indicia of theconformational disruption occasioned by the substitution), fibrinogenclotting (a measure of procoagulant activity), protein C activation (ameasure of anticoagulant activity), the ratio of protein C activation tofibrinogen clotting activity, and heparin-dependent AT-III inhibition (ameasure of the ability of the NP to withstand inactivation by AT-III inthe presence of heparin). Expression in our system of NPs Mt3 and Mt37ccould not be detected. TABLE 1 Sp Sp Heparin- Residues Chymo- AmidolyticAc Amidolytic Ac Fibrinogen Protein C dependent NPs Substitutedtrypsinogen Slot Blot Western Blot Clotting (FC) Activation (PA) ATIIIInhibition Unit with Alanine Numbering % of wild type % of wild type %of wild type % of wild type PA/FC % Residual Activity Wild Type 99.09100.00 100.00 100.00 1.00 18.15 Mt1 S4a, E6a, D8a 1E, 1C, 1A 281.5858.32 165.45 93.07 0.56 Mt2 K17a, K18a, S19a 9, 10, 11 104.78 99.2784.78 96.94 1.14 Mt3 K23a, R26a, E27a 14A, 14D, 14E — — — — — Mt3a K23a14A 111.94 91.46 110.54 87.39 0.79 Mt3b R26a 14D 96.42 79.02 92.48 99.361.07 Mt3c E27a 14E 61.87 70.23 46.34 59.74 1.29 Mt4 E30a, D34a 14H, 14L96.97 93.86 80.08 102.71 1.28 Mt5 E3, D6 18, 21 101.61 93.44 85.57 97.231.14 Mt6 R20 35 136.11 92.16 79.95 67.73 0.85 Mt7 K21 36 92.13 90.7730.91 14.24 0.46 Mt8 S22, Q24, E25 37, 38, 39 129.96 90.30 158.49 11.850.07 Mt8a S22 37 109.65 69.36 74.82 47.90 0.64 Mt8b Q24 38 122.15 70.13135.51 12.54 0.09 Mt8c E25 39 101.06 81.87 119.98 104.42 0.87 Mt9 D35 49233.65 92.52 133.46 112.81 0.85 Mt10 W50, D51 60D, 60E 43.73 76.57 12.6824.98 1.97 Mt10a W50 60D 49.37 71.40 5.15 35.56 6.90 Mt10b D51 60E110.80 75.17 96.50 69.08 0.72 Mt11 K52 60F 122.22 85.22 38.17 134.703.53 Mt12 N53, T55 60G, 60I 76.22 70.51 42.60 99.58 2.34 Mt13 N57, D5862, 63 83.94 90.14 47.90 71.77 1.50 Mt13a N57 62 96.64 92.93 75.17100.14 1.33 Mt13b D58 63 93.34 71.98 58.11 87.75 1.51 Mt14 K65 70 110.97100.76 1.85 5.64 3.04 Mt14.5 H66 71 55.45 103.45 2.00 12.30 6.14 Mt15R68 73 99.53 78.01 20.25 34.12 1.68 Mt16 T69 74 120.84 107.33 77.0458.26 0.76 Mt17 R70A 75 108.39 93.34 77.51 14.01 0.18 Mt17a R70E 75105.69 95.31 76.86 63.29 0.82 Mt17b R68, R70 73, 75 109.32 69.25 6.7013.31 1.99 Mt18 Y71 76 87.95 102.03 0.31 5.28 17.11 Mt19 R73 77A 91.7499.34 16.65 26.38 1.58 Mt20 N74, K77 78, 81 127.70 124.90 10.59 12.921.22 Mt20a N74 78 94.97 86.47 59.33 82.47 1.39 Mt20b K77 81 80.46 82.4727.05 12.71 0.47 Mt21 E82, K83 86, 87 124.83 139.61 71.83 62.45 0.87Mt22 R89, R93, E94 93, 97, 97A 142.67 95.11 54.92 61.10 1.11 59.24Mt22.5 R98 101 186.13 46.67 124.06 62.50 0.50 Mt23 K106, K107 109, 110107.35 102.01 13.15 31.42 2.39 Mt23a K106 109 86.92 93.24 50.25 72.351.44 Mt23b K107 110 106.96 92.38 46.93 44.92 0.96 Mt24 D113 116 122.3690.97 80.15 88.41 1.10 Mt25 D122, R123, E124 125, 126, 127 92.57 71.4784.21 84.44 1.00 Mt26 S128, Q131 129B, 131 75.74 109.30 85.40 89.80 1.05Mt27 K145, T147, W148 145, 147, 148 111.11 98.07 65.81 84.40 1.28 Mt28T149, N151 149, 149B 81.88 98.90 74.36 76.63 1.03 Mt29 K154 149E 88.5787.07 118.76 95.47 0.80 Mt30 S158 153 86.86 92.30 97.96 97.50 1.00 Mt31E169, K174, D175 164, 169, 170 111.22 205.21 43.97 69.56 1.58 32.86 Mt32R178, R180, D183 173, 175, 178 61.62 58.44 90.35 102.37 1.13 56.52 Mt33D193, K196 186A, 186D 99.65 108.75 28.87 60.27 2.09 Mt34 E202 192 40.3325.40 37.22 93.81 2.52 Mt35 N216, N217 204B, 205 177.29 92.87 41.6477.56 1.86 Mt36 E229, R233, D234 217, 221, 222 0.75 0.00 0.00 Mt36a E229217 42.70 23.73 0.58 13.22 22.91 Mt36b R233 221 75.90 83.93 2.31 25.1610.87 Mt36c D234 222 51.82 75.33 27.37 59.64 2.18 Mt37 R245, K248, Q251233, 236, 239 0.73 0.00 0.00 Mt37a R245 233 135.96 26.22 72.57 70.390.97 Mt37b K248 236 143.41 73.41 76.89 76.86 1.00 Mt37c W249 237 — — — —— Mt37d Q251 239 105.24 96.55 60.45 68.79 1.14 Mt38 K252, D255, Q256240, 243, 244 92.80 94.98 65.07 63.74 0.98

[0053] The most salient parameter of Table 1 is the PA/FC ratio. Themore this ratio varies from 1.0, the greater the segregation ofprocoagulant and PC activating properties in the mutants. Those with thehighest arithmetic value are particularly dedicated to PC activation,while those with the lowest value are dedicated to procoagulantfunction. However, it is within the scope of this invention tosubstitute additionally any amino acid residue other than alanine at theTable 1 sites, e.g. one of the 20 naturally occurring residues disclosedbelow. For optimal anticoagulant therapeutic utilities the PC activatingcapabilities of the NP should be at least about 5%, ordinarily 10%-30%,of reference sequence thrombin, notwithstanding the PA/FC ratio, so thatone can ensure that sufficient activity of the enzyme will be present toexert a physiological or diagnostic effect. In addition, the absolutefibrin clotting activity of the PCA ideally should be less than about10% of reference thrombin, generally less than about 5% and ordinarilyless than about 3% and preferably less than about 1% of wild-type, tohelp ensure clinical safety. It is not necessary that the NP retain thesame level of protein C activating activity as reference sequencethrombin because lower potency is readily overcome in therapy by simplyadministering more of the enzyme. In this regard the E229 and R233 PCAsare particularly attractive.

[0054] In addition to the alanine substitutions shown in Table 1, it iswithin the scope of this invention to substitute other residues into thethrombin reference sequence. The residues inserted into the sequencegenerally are naturally occurring amino acids, commonly G, A, Y, V, L,I, S, T, D, E, Q, C, M, N, F, P, W, K, R or H (using conventional singleletter code; EP 323,149). Suitable residues for insertion also includehydroxyproline, beta-hydroxyaspartic acid, gamma-carboxyglutamic acid,hydroxylysine or norleucine, to be employed as alternatives to theirnamesakes.

[0055] These substitutions may be conservative in that the substitutingresidue will be bear structural or functional similarity to thesubstituted residue. Other substitutions will be less conservative inthat they constitute an exchange between different structural orfunctional classes of residues. For the purposes herein, these classesare as follows: 1. Electropositive: R, K, H; 2. Electronegative: D, E;3. Aliphatic: V, L, I, M; 4. Aromatic: F, Y, W; 5. Small: A, S, T, G, P,C; 6. Charged: R, K, D, E, H; 7. Polar: S, T, Q, N, Y, H, W; and 8.Small Hydrophilic: C, S, T. Intergroup substitutions generally will havegreater effects on protein function than conservative (intraclass)substitutions. Thus, it is particularly within the scope of thisinvention to introduce conservative substitutions into the Table 1 or 1bsites and, if the results are not satisfactory, to introducenon-conservative substitutions at the sites. Typically, however,proline, glycine, and cysteine substitutions or insertions into thesequence are not favored.

[0056] An object of this invention is to obtain NPs that are minimallyimmunogenic or non-immunogenic in humans. In this regard, substitutionsor insertions of K or R residues into the sequence are not favored.

[0057] Substitutions preferably are made at Table 1 sites where alaninesubstitution results in a PA/FC ratio that is greater than 2.0 or lessthan 0.5 (W50, K52, D58, K65, H66, Y71, N74, E202, E229, R233, D234,K21, Q24, R70, R98 and K77). Four of these sites were selected forsaturation mutagenesis (E229, R233, W50 and K52). In addition, doubleand triple mutations variously were introduced into sites W50, K52,R233, E229, E202, K106, K107, D193 and K196. These NPs were prepared andexpressed in recombinant cell culture in the same fashion as the Table 1mutants, and then assayed for expression levels, amidolytic activity,aPC and fibrinogen clotting activity. The results are shown in Table 1a(“INF”=approaches infinity; blank NPs have not yet been characterized).TABLE 1a Expression Sp Amidolytic Ac Protein C Fibrinogen NPs LevelWestern Blot Activation (PA) Clotting (FC) PA/FC Unit % of wild type %of wild type % of wild type % of wild type Ratl S205A 83.50 0.26 0.010.00 WT 100.00 100.00 100.00 100.00 1.00 W50A/K52A 24.50 94.90 77.576.87 11.28 W50A/E229A 55.50 7.28 1.45 0.00 INF W50A/R233A 3.90 267.50−0.13 0.00 0.00 K52A/E202A K52A/E229A 4.30 41.87 6.79 K52A/R233A 14.4094.29 18.50 0.00 INF K52A/K106A/K107A 2.20 171.02 15.92 K52A/D193A/K196AE229A/R233A 45.00 10.90 0.57 0.00 INF E229A 62.50 33.44 5.25 0.33 15.81E229C 0.00 E229D 45.00 185.00 10.21 0.00 INF E229F 11.00 40.57 2.89 0.00INF E229G 100.00 3.48 −2.97 0.00 0.00 E229H E229I E229K 16.00 23.29 3.95E229L 22.50 79.20 44.62 20.98 2.13 E229M E229N 61.00 101.83 12.19 E229P113.00 6.26 1.84 0.00 INF E229Q E229R E229S 30.00 52.91 25.83 0.53 48.75E229T 16.00 56.36 14.37 5.32 2.70 E229V 16.00 58.25 9.16 12.62 0.73E229W 20.00 34.57 29.05 0.00 INF E229Y 8.50 38.17 33.43 0.00 INF R233A81.00 113.71 55.04 3.04 18.11 R233C 52.00 58.06 6.38 0.00 INF R233D75.00 84.93 15.20 0.00 INF R233E 52.00 229.46 67.61 1.04 65.10 R233F100.00 59.91 9.24 0.00 INF R233G 50.00 110.77 10.25 0.87 11.76 R233H96.00 94.96 25.15 4.59 5.48 R233I 77.50 73.22 31.36 6.27 5.00 R233KR233L 96.00 79.47 10.82 7.15 1.51 R233M 200.00 46.78 10.04 1.15 8.69R233N 52.00 242.15 80.61 1.06 76.12 R233P 50.00 5.49 −0.74 0.00 0.00R233Q 96.00 91.05 14.90 4.96 3.00 R233S 80.00 94.86 12.87 0.90 14.33R233T 80.00 98.91 6.95 1.46 4.76 R233V 50.00 122.85 28.02 23.15 1.21R233W 0.00 R233Y 106.00 27.46 −0.83 0.00 0.00 W50A 53.60 123.04 50.845.17 9.83 W50C 25.00 48.63 20.38 0.00 INF W50D 32.46 220.03 9.99 7.641.30

[0058] TABLE 1 Sp Sp Heparin- Residues Chymo- Amidolytic Ac AmidolyticAc Fibrinogen Protein C dependent NPs Substituted trypsinogen Slot BlotWestern Blot Clotting (FC) Activation (PA) ATIII Inhibition Unit withAlanine Numbering % of wild type % of wild type % of wild type % of wildtype PA/FC % Residual Activity Wild Type 99.09 100.00 100.00 100.00 1.0018.15 Mt1 S4a, E6a, D8a 1E, 1C, 1A 281.58 58.32 165.45 93.07 0.56 Mt2K17a, K18a, S19a 9, 10, 11 104.78 99.27 84.78 96.94 1.14 Mt3 K23a, R26a,E27a 14A, 14D, 14E — — — — — Mt3a K23a 14A 111.94 91.46 110.54 87.390.79 Mt3b R26a 14D 96.42 79.02 92.48 99.36 1.07 Mt3c E27a 14E 61.8770.23 46.34 59.74 1.29 Mt4 E30a, D34a 14H, 14L 96.97 93.86 80.08 102.711.28 Mt5 E3, D6 18, 21 101.61 93.44 85.57 97.23 1.14 Mt6 R20 35 136.1192.16 79.95 67.73 0.85 Mt7 K21 36 92.13 90.77 30.91 14.24 0.46 Mt8 S22,Q24, E25 37, 38, 39 129.96 90.30 158.49 11.85 0.07 Mt8a S22 37 109.6569.36 74.82 47.90 0.64 Mt8b Q24 38 122.15 70.13 135.51 12.54 0.09 Mt8cE25 39 101.06 81.87 119.98 104.42 0.87 Mt9 D35 49 233.65 92.52 133.46112.81 0.85 Mt10 W50, D51 60D, 60E 43.73 76.57 12.68 24.98 1.97 Mt10aW50 60D 49.37 71.40 5.15 35.56 6.90 Mt10b D51 60E 110.80 75.17 96.5069.08 0.72 Mt11 K52 60F 122.22 85.22 38.17 134.70 3.53 Mt12 N53, T5560G, 60I 76.22 70.51 42.60 99.58 2.34 Mt13 N57, D58 62, 63 83.94 90.1447.90 71.77 1.50 Mt13a N57 62 96.64 92.93 75.17 100.14 1.33 Mt13b D58 6393.34 71.98 58.11 87.75 1.51 Mt14 K65 70 110.97 100.76 1.85 5.64 3.04Mt14.5 H66 71 55.45 103.45 2.00 12.30 6.14 Mt15 R68 73 99.53 78.01 20.2534.12 1.68 Mt16 T69 74 120.84 107.33 77.04 58.26 0.76 Mt17 R70A 75108.39 93.34 77.51 14.01 0.18 Mt17a R70E 75 105.69 95.31 76.86 63.290.82 Mt17b R68, R70 73, 75 109.32 69.25 6.70 13.31 1.99 Mt18 Y71 7687.95 102.03 0.31 5.28 17.11 Mt19 R73 77A 91.74 99.34 16.65 26.38 1.58Mt20 N74, K77 78, 81 127.70 124.90 10.59 12.92 1.22 Mt20a N74 78 94.9786.47 59.33 82.47 1.39 Mt20b K77 81 80.46 82.47 27.05 12.71 0.47 Mt21E82, K83 86, 87 124.83 139.61 71.83 62.45 0.87 Mt22 R89, R93, E94 93,97, 97A 142.67 95.11 54.92 61.10 1.11 59.24 Mt22.5 R98 101 186.13 46.67124.06 62.50 0.50 Mt23 K106, K107 109, 110 107.35 102.01 13.15 31.422.39 Mt23a K106 109 86.92 93.24 50.25 72.35 1.44 Mt23b K107 110 106.9692.38 46.93 44.92 0.96 Mt24 D113 116 122.36 90.97 80.15 88.41 1.10 Mt25D122, R123, E124 125, 126, 127 92.57 71.47 84.21 84.44 1.00 Mt26 S128,Q131 129B, 131 75.74 109.30 85.40 89.80 1.05 Mt27 K145, T147, W148 145,147, 148 111.11 98.07 65.81 84.40 1.28 Mt28 T149, N151 149, 149B 81.8898.90 74.36 76.63 1.03 Mt29 K154 149E 88.57 87.07 118.76 95.47 0.80 Mt30S158 153 86.86 92.30 97.96 97.50 1.00 Mt31 E169, K174, D175 164, 169,170 111.22 205.21 43.97 69.56 1.58 32.86 Mt32 R178, R180, D183 173, 175,178 61.62 58.44 90.35 102.37 1.13 56.52 Mt33 D193, K196 186A, 186D 99.65108.75 28.87 60.27 2.09 Mt34 E202 192 40.33 25.40 37.22 93.81 2.52 Mt35N216, N217 204B, 205 177.29 92.87 41.64 77.56 1.86 Mt36 E229, R233, D234217, 221, 222 0.75 0.00 0.00 Mt36a E229 217 42.70 23.73 0.58 13.22 22.91Mt36b R233 221 75.90 83.93 2.31 25.16 10.87 Mt36c D234 222 51.82 75.3327.37 59.64 2.18 Mt37 R245, K248, Q251 233, 236, 239 0.73 0.00 0.00Mt37a R245 233 135.96 26.22 72.57 70.39 0.97 Mt37b K248 236 143.41 73.4176.89 76.86 1.00 Mt37c W249 237 — — — — — Mt37d Q251 239 105.24 96.5560.45 68.79 1.14 Mt38 K252, D255, Q256 240, 243, 244 92.80 94.98 65.0763.74 0.98

[0059] The data in Table la were obtained as provided in Table 1, andthat the activities (protein C activation and fibrinogen clotting) werenormalized by amidolytic activity, except that in Table 1a when thespecific amidolytic activity of NP was less than 75% of correspondingwild type thrombin, the NP activity values for protein C activation andfibrinogen clotting were corrected by multiplying by the correspondingspecific amidolytic activity of the NP (expressed as % of wild type). Itwill be noted that the numerical values for PA and FC activity differbetween Tables 1 and la for NPs appearing in both Tables. This is theresult of two factors. First, the Table 1a results generally representthe outcome of only 1 or 2 replicate assays, whereas those of Table 1are based on up to 4 replicates. Since the coefficient of variation ofthe PA and FC assays is in the range of about 10-20%, the Table 1aresults can be expected to vary from those of Table 1 for the same NPs.Second, the Table 1a results were corrected for protein concentrationbased on Western blotting if the amidolytic activity was less than 75%of wild-type thrombin, as described above. This allows for a moremeaningful comparison among the various NPs in Table 1a since theresults are corrected to the same protein concentration as determined byWestern blot. Correction was largely unnecessary with the Table 1 NPsbecause, for the most part, the alanine mutants retained most of theproteolytic activity of reference sequence thrombin.

[0060] Particularly remarkable PCAs were identified in Table 1a; thedata indicates retention of at least some aPC activity but essentiallycomplete elimination of detectable FC activity in our assay (600 secondswithout clotting). These were the double mutants W50A, E229A and E229A,R233A; and the single mutants E229D (which remarkably only differs fromthe wild type enzyme by a single methylene group), E229F, E229S, E229W,E229Y, R233E, R233G, R233M, R233N, R233S, W50E, W50K, W50C, and K52C.

[0061] The Table 1 and 1a PCAs are only representative of NPs in whichthrombin residues are mutated in order to produce particularly safe andpotent PCAs. Other such PCAs are readily identified by the methodsdescribed herein or other methods apparent to the ordinary artisan. Forexample, multiple sites of variation are selected by additivity ofactivities, and other promising sites identified by alanine scanning aresubjected to saturation mutagenesis to select the optimal modification.Since it is apparent that other NPs having the desired procoagulant andanticoagulant properties can be successfully identified, it would bemerely a matter of routine experimentation to identify and isolate them.

[0062] It is apparent from Tables 1 and 1a that E229 and R233 are keyresidues for PCAs. The modification of thrombin residues in Van derWaals contact with E229 or R233, residues near E229 or R233 and residuesin contact with the domain containing R233 and the loop that containsE229 may affect fibrinogen clotting and protein C activation in a manneranalogous to the direct substitution of E229 or R233 by causing theposition or orientation of E229 or R233 to change, or by disruptingintramolecular interactions normally associated with E229 or R233. Inorder to identify residues whose substitution might mimic thesubstitution of E229, the residues in close proximity to E229 in thethree-dimensional structure of thrombin (Bode, W. et al, EMBO J.8:3467:3475 (1989)) were mapped. Residues whose Cα. was within a 10 Åsphere surrounding the Cα of E229 are listed in Table 1b below. TABLE 1bDistance Cα to Cα E229 Mutation Residue (Å) in Table 1 PA/FC E146 10.06— S176 9.93 — T177 8.28 — R178 10.31 Mt 32 1.13 I179 10.03 — D199 10.91— A200 10.90 — C201 9.74 — E202 10.06 Mt 34 2.52 W227 7.27 — G228 3.82 —G230 3.73 — C231 6.69 — D232 8.88 — R233 7.94 Mt 36b 10.87 D234 10.64 Mt36c 2.18 G235 10.41 — K236 6.96 — Y237 7.75 — G238 7.88 — F239 9.32 —

[0063] One of the sites independently identified by this analysis isR233, which is demonstrated by the results reported in Table 1a to playa significant role in PCA specificity. Three other sites had beenidentified in the original screen reported in Table 1 and all three ofthem yielded a PA/FC ratio greater than 1, again confirming theinstrumental role of this domain in segregating thrombin's substratespecificity towards aPC activity. Accordingly, it is within the scope ofthis invention to prepare NPs in which a residue has been insertedadjacent to, substituted for or deleted at any one or more of theresidues scheduled in Table 1b. In particular, residues lying within 10Angstroms of the E229 Cα or R233 Cα are of particular interest, andthose that are within 8 Angstroms even more so. However, C201 and C231are not preferred sites as they are paired to form a disulfide bond innative thrombin. G228, G230, G235 and G238 also are not preferred.Accordingly, preferred Table 1b residues for substitution, insertion ordeletion are S176, T177, W227, D232, K236, Y237, and G239. Substitutionsfor the Table 1b residues are preferred over deletions or insertions,and are made with class members other than the one to which the nativeresidue belongs, or the substitutions may be by other members of thenative residue's class. In general, however, cysteine, proline andglycine will not be substituted in place of any of the Table 1bresidues. The residues within 10 Angstroms of R233 will overlap many ofthe E229 proximal residues. Additional ones that do not overlap arereadily identified using the three dimensional structure of Bode (opcit).

[0064] PCA polypeptides generally are polypeptides in which residues atone or more of the following thrombin sites have been substituted forthe corresponding reference sequence residues: K52, K65, Y71, N74, W50,D58, H66, E202, E229, D234 and R233. The residues K52, K106, K107, K196and/or K65 independently preferably are substituted with a naturallyoccurring amino acid residue, e.g., G, A, V, L, I, S, T, D, N, E, Q, C,M, F, Y, P, W, R or H. K52 ordinarily is substituted with cysteine,glycine, alanine, glutamic acid, aspartic acid, asparagine or glutamine,in some instances with any naturally occurring amino acid residue otherthan glutamic acid, or in other embodiments, with a residue other thanglutamic acid or aspartic acid, while K65, K106, K107 and/or K196typically are substituted with alanine, glutamic acid, aspartic acid,asparagine, glutamine, arginine or histidine.

[0065] Y71 is substituted preferably with a naturally occurring aminoacid residue, e.g., G, A, V, L, I, S, T, D, E, Q, C, M, N, F, P, W, K, Ror H, typically phenylalanine, threonine, serine, isoleucine, ortryptophan.

[0066] N74 is substituted preferably with a naturally occurring aminoacid residue, e.g., G, A, V, L, I, S, T, D, E, Q, C, M, F, P, W, K, R,or H, typically alanine, glutamine, glutamic acid, or aspartic acid,

[0067] W50 is substituted preferably with a naturally occurring aminoacid residue, e.g., G, A, V, L, I, S, T, D, E, Q, C, M, F, P, N, K, R,Y, or H, typically cysteine, alanine, phenylalanine, lysine, arginine orhistidine.

[0068] D58, D193 and/or D234 independently are substituted preferablywith a naturally occurring amino acid residue, e.g., G, A, V, L, I, S,T, W, E, Q, C, M, F, P, N, K, R, Y, or H, typically alanine, glutamicacid, glutamine, asparagine, tyrosine, threonine or serine.

[0069] H66 is substituted preferably with a naturally occurring aminoacid residue, e.g., G, A, V, L, I, S, T, W, D, Q, C, M, F, P, N, K, Y, Eor R, typically alanine, lysine, asparagine, glutamine, threonine,histidine, or serine.

[0070] E202 and/or E229 independently are substituted preferably with anaturally occurring amino acid residue, e.g., G, A, V, L, I, S, T, W, D,Q, C, M, F, P, N, K, R, Y, or H, typically alanine, aspartic acid,phenylalanine, tryptophan, glutamine, asparagine, aspartic acid,tyrosine or serine.

[0071] R233 is substituted preferably with a naturally occurring aminoacid residue, e.g., G, A, V, L, I, S, T, W, D, Q, C, M, F, P, N, K, E,Y, or H, typically alanine, lysine, asparagine, glutamine, glutamicacid, threonine, histidine or serine.

[0072] NPs representing combinations of the foregoing are within thescope of this invention. 2, 3, 4, 5, or more substitutions from theforegoing are introduced into thrombin as defined herein. The results ofindividual amino acid substitutions are generally additive except whenthe residues interact with each other directly or indirectly. We expectadditional PCAs to be obtained from multiple substitutions atcombinations of the 11 key residues described above (W50, K52, D58, K65,H66, Y71, N74, K106, K107, D193, K196, E202, E229, R233, D234). They arereadily screened using the same procedures described below in order toidentify those having improved properties, in particular an enhancedreduction in procoagulant activity relative to anticoagulant activity.They include for example E229,R233,D234; E229,R233; E229,D234;R233,D234; E229,K52; R233,K52; D234,K52; E229,R233,K52; E229,D234,K52;R233,D234,K52; E229,R233,D234,K52; W50,D58; W50,K52; W50,E229; W50,R233;W50,D234; W50,E229,R233,D234; W50,E229,R233; W50,E229,D234;W50,R233,D234; W50,E229,K52; W50,R233,K52; W50,D234,K52;W50,E229,R233,K52; W50,E229,D234,K52; W50,R233,D234,K52;W50,E229,R233,D234,K52; E202,E229,R233,D234; E202,E229,R233;E202,E229,D234; E202,R233,D234; E202,E229,K52; E202,E233,K52;E202,D234,K52; E202,E229,R233,K52; E202,E229,D234,K52;E202,R233,D234,K52; E202,E229,R233,D234,K52; E202,W50,K52;E202,W50,E229; E202,W50,R233; E202,W50,D234; E202,W50,R233,D234;E202,W50,E229,R233; E202,W50,E229,D234; E202,W50,R233,D234;E202,W50,K52; E202,W50,E229; E202,W50,R233; E202,W50,D234; W50,E202;E202,K52; E202,E229; E202,R233; E202,D234; D58,K52: D58,E229; D58,R233;D58,D234; D58,W50; D58,E202; D58,K52,E229; D58,K52,R233; D58,K52,D234;D58,K52,W50; D58,K52,E202; D58,E229,R233; D58,E229,D234; D58,E229,W50;D58,E229,E202; D58,R233,D234; D58,R233,W50; D58,R233,E202; D58,D234,W50;D58,R233,E202; D58,W50,E202; D58,W50,R233; and D58,W50,D234.

[0073] Exemplary embodiments of multiple-substituted NPs fall within thefollowing (the class of substitution is designated by the class numberabove, e.g. W50.3 means W50 substituted with any of V, L, I or M):W50.1,.2,.3,.5,.6,.7 or .8,K52.2,.3,.4,.5,.6,.7 or .8;W50.1,.2,.3,.5,.6,.7 or .8,D58.1,.3,.4,.5,.6,.7 or .8;W50.1,.2,.3,.5,.6,.7 or .8,H66.2,.3,.4,.5,.6,.7 or .8;W50.1,.2,.3,.5,.6,.7 or .8,Y71.1,.2,.3,.5,.6,.7 or .8;W50.1,.2,.3,.5,.6,.7 or .8,E229.1,.3,.4,.5,.6,.7 or .8;K52.2,.3,.4,.5,.6,.7 or .8,D58.1,.3,.4,.5,.6,.7 or .8;K52.2,.3,.4,.5,.6,.7 or .8,H66.2,.3,.4,.5,.6,.7 or .8;K52.2,.3,.4,.5,.6,.7 or .8,Y71.1,.2,.3,.5,.6,.7 or .8;K52.2,.3,.4,.5,.6,.7 or .8,R233.2,.3,.4,.5,.6,.7 or .8;D58.1,.3,.4,.5,.6,.7 or .8,H66.2,.3,.4,.5,.6,.7 or .8;D58.1,.3,.4,.5,.6,.7 or .8,Y71.1,.2,.3,.5,.6,.7 or .8;D58.1,.3,.4,.5,.6,.7 or .8,E202.1,.3,.4,.5,.6,.7 or .8;D58.1,.3,.4,.5,.6,.7 or .8,R33.2,.3,.4,.5,.6,.7 or .8;H66.2,.3,.4,.5,.6,.7 or .8,Y71.1,.2,.3,.5,.6,.7 or .8;H66.2,.3,.4,.5,.6,.7 or .8,E229.1,.3,.4,.5,.6,.7 or .8;H66.2,.3,.4,.5,.6,.7 or .8,R33.2,.3,.4,.5,.6,.7 or .8;Y71.1,.2,.3,.5,.6,.7 or .8,E229.1,.3,.4,.5,.6,.7 or .8; andY71.1,.2,.3,.5,.6,.7 or .8,R233.2,.3,.4,.5,.6,.7 or .8.

[0074] Particular multiple-substituted NPs are W50F,Y or H,D58E,N or Q;W50F,Y or H,H66F,Y or W; W50F, Y or H,Y71F, H or W; W50F, Y or H,E229D,N or Q; W50F,Y or H,R233K or H; D58E, N or Q,H66F, Y or H; D58E, N orQ,Y71F, H or W; D58E, N or Q,E229D, N or Q; D58E, N or Q,R233K or H;H66F, Y or W,Y71F, H or W; H66F, Y or W,E229D, N or Q; H66F, Y orW,R233K or H; Y71F, H or W,E229D, N or Q; Y71F, H or W,R233K or H;E229D, N or Q,R233K or H; W50A,K52A; W50A,E229A; W50A,R233A;K52A,K106A,K107A; K52A,D193A,K196A; K52A,E202A; K52A,E229A; K52A,K233A;E229L,R233E; E229L,R233N; E229L,W50K; E229L,K52W; E229A,W50L;E229L,W50M; R233E,W50G; R233E,W50K; R233E,W50K; R233E, W50L; R233E,W50M;R233E,W50R; R233E,W50T; R233E,K52W; W50L,K52A; W50L,K52W; G230C,C231G;C231D,D232C; E202C,C201E; C201A,A200C; and E229A,R233A.

[0075] Further exemplary multiple-substituted NPs are selected from thefollowing: K52G,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, Kor H; K52A,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K orH; K52V,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52L,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52I,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52S,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52T,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52D,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52N,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52E,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52Q,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52M,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52F,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52Y,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52P,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52W,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52R,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;K52H,E229G, A, V, I, L, S, T, D, N, C, Q, M, F, Y, P, W, R, K or H;W50G,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50A,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50V,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50L,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50I,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50S,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50T,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50D,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50N,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50E,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50Q,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50M,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50F,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50Y,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50P,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50K,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50R,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50H,E229G, A, V, I, L, S, T, D, N, Q, C, M, F, Y, P, W, R, K or H;W50G,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50A,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50V,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50L,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50I,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y,P,N,K, E orH;W50S,R233G,A,V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50T,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50D,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50N,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50E,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50Q,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50M,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50F,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50Y,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50P,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50K,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50R,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;W50H,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229G,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229A,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229V,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229L,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229I,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229S,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229T,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229D,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229N,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229Q,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229M,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229F,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229Y,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229P,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229K,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229R,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H;E229H,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H; andE229W,R233G, A, V, I, L, S, T, W, D, Q, C, M, F, Y, P, N, K, E or H.

[0076] Included within the scope of this invention are NPs having one ormore amino acids inserted immediately adjacent to a thrombin amino acidat any of the designated Table 1, 1a or 1b positions in the referencesequence. Insertional NPs generally will have a polypeptide structurecomprising the sequence NH₂—PP-A-(X)_(n1)—B—PP—COOH, wherein X is theinserted residue(s) (which may be the same or different), n1 is aninteger, either A or B are the designated residue sites for insertionand PP represents the remainder of the NP or a bond at the N or Cterminus of the NP. Examples include K52AA, R233RA, K52KA, K52AK,E229AE, E229EW, E229WE, E229EY, E229YE, G228GE, G228GA, G228GS, G228GAA,G228GAE, G228GSE, G230GA, G230GS, G230GAA, and E229EA (reading in the Nto C terminal direction). Insertions typically are found within about 10Angstroms of E229 and adjacent to any Table 1b residue. Insertionsinclude thrombin or non-thrombin polypeptides ranging from 1 to about1000 or more residues, although these are typically introduced at the Nor C-terminal ends of the NP A and/or B chains. These polypeptidesinclude prothrombin sequences or fragments thereof (whether from humansor animals), antigenic sequences for immunoaffinity purification of theNP products from cell culture, signal sequences and the like as are morefully described below.

[0077] Also included within the scope of this invention are NPs in whicha glycosylation site is introduced or removed from the referencesequence, whether by substitution, insertion or deletion of one or moreamino acid residues. Such changes will result in the installation orremoval of the sequence NXS or NXT, where X can be any residue. Thus,asparagine can be substituted for any residue located 2 residuesN-terminal to serine or threonine to introduce a glycosylation site.Alternatively, the single glycosylation site of wild-type thrombin atN53 can be omitted by substituting N53 with any residue, deleting F54,substituting any residue other than serine for T55 or inserting at leastone residue between N53 and T55.

[0078] Also included within the scope of this invention are deletionalNPs, i.e., NPs in which one or more amino acid residues of the referencesequence have been removed at a designated site, whereby flankingresidues are now joined by a peptide bond in the ordinary fashion. Anyof the sites set forth in Tables 1 or 1b are suitable for deletion,although it generally is not preferred to delete P, C or G residues. Inaddition, the thrombin A chain in its entirety optionally is deleted insome NP embodiments. In embodiments of this invention, deletions aremade within about 10 Angstroms of E229 and include any of the residuesin Table 1b, preferably A200, E202, Y237, I179, R178, E146, as well asS226.

[0079] Typically, deletions or insertions are relatively small, on theorder of 1 to 10 residues and generally no more than 2, althoughdeletions or insertions can be quite large if they are not in portionsof the reference sequence required for procoagulant or anticoagulantactivity as the case may be, or the additional sequence is to be removedat some point during post-translational or post-recovery processing. Thenumber of residues that are deleted or inserted in part will depend uponwhether or not they are found in secondary structural components such ashelices or sheets (whereupon only 1 or, preferably 2 residues areinserted or deleted), or are in less structurally confined domains suchas loops, where larger numbers of residues may be deleted or insertedwithout unduly perturbing the structure of thrombin.

[0080] Also included within the scope of this invention are NPs havingcombinations of deletions, insertions and/or substitutions. Typically, adeletion of a single residue will be accompanied by an insertion within1 to about 3 residues of the deletion site; deletions of larger domainsnot necessary for procoagulant or aPC activity, as the case may be, neednot be accompanied by an insertion. The thrombin A chain optionally isdeleted from NPs, in which case the B chain cysteine residue thatordinarily forms a disulfide bond with the A chain (C119) is substitutedor deleted from the B chain. Typical substitutions at C119would be anyof R, G, A, V, I, L, S, T, W, D, Q, M, F, Y, P, N, K, E or H, butordinarily would be S, M or A. Most insertions employed for purposes offacilitating the expression or recovery of NPs will naturally beaccompanied by other modifications in the reference sequence that conferthe desired properties on the NPs, e.g. as to anticoagulant orprocoagulant effect.

[0081] The NPs of this invention may be subject to post-translationalcovalent modification, e.g. deamidation of asparagine or glutamine, oroxidation of cysteine residues, and absent or variant glycosylation atN53 depending upon the host cell used to express the variant. NPscontaining such modifications are included within the scope of thisinvention. If N53 is glycosylated, it preferably is glycosylated withcarbohydrates characteristic of mammalian cells, although it also maybear fungal (such as yeast) glycosylation patterns. Glycosylationcharacteristic of expression of the NP from fibroblast, kidney, lung,skin, neural, liver or bone marrow cells or cell lines, or of anymammalian cell line such as CHO or embryonic kidney cells, isacceptable.

[0082] One major mechanism of thrombin clearance in vivo is theformation of a thrombin-AT-III complex, which is largely dependent oncell surface heparin-like molecules. We expect that mutating thrombin'sheparin-binding site will prevent heparin binding and thereby prolongthe plasma half-life of the protein. Clinically, this will reduce theamount of protein required to achieve an anti-thrombotic effect. In thisembodiment the heparin binding site is mutagenized so that the NP nolonger is capable of substantially binding to heparin. This isaccomplished by deletion, substitution or insertion of one or moreresidues in at least the known heparin binding domain (including R89,R180, R245, K248 and K252). Resistant NPs include substitutions orinsertions that result in the introduction of a novel O- or N-linkedglycosylation site (NXS/T) into the binding region (for example R180N).

[0083] Table 1 shows that two mutants, each involving triple alaninesubstitutions, were resistant to heparin-mediated inhibition by AT-III,exhibiting greater than 50% of fibrinogen clotting activity in thepresence of heparin compared to 18% for wild-type thrombin. One of thesemutants, involving the simultaneous substitution of R178, R180 and D183,displayed no reduction in procoagulant or anticoagulant activity.Optimal mutants are identified by screening for those that are mostresistant to heparin-mediated inhibition, particularly in the presenceof antithrombin III. Mutations of this type optionally are combined withvariations described above, e.g., those that display a reduction inprocoagulant activity relative to anticoagulant activity. Such NPs areadministered in combination with heparin to achieve a potentanticoagulant effect where the anticoagulant pathway is activated by theNP and the procoagulant activity of the endogenous thrombin is inhibitedby heparin-mediated inhibition by AT-III.

Preparation and Selection of NPs

[0084] Optimal NPs which exhibit modulate fibrinogen clotting or aPC,reduced heparin inhibition, modified ability to cleave the plateletthrombin receptor, and other desired properties, are identified byscreening candidates against the relevant in vitro assays as describedherein, then testing in animals as desired followed by determination ofefficacy in thrombosis or bleeding models. Exogenous aPC may be used asa positive control. The selected NPs optionally then are directly testedin animal models in which aPC infusion has been shown to be efficacious,e.g. in rabbit, guinea pig, or baboon septic shock or arterialthrombosis models. Additionally, the anticoagulant potency of such NPscan be tested in cardiopulmonary bypass models. These assays areconventional and widely known in the art. It would not require undueexperimentation to make and test other NPs than those specificallydisclosed herein.

[0085] The NPs of this invention are readily prepared by methods knownin the art. In general, nucleic acid encoding the NP is prepared by PCR,in vitro synthesis, cloning or combinations of the three, including ifnecessary the site directed mutagenesis of thrombin-encoding nucleicacid. It is expressed in in vitro systems or in recombinant host cells.One method for expression is ribosome based synthesis using dedicatedtRNAs (Benner, “TIBTECH” 12:158-163 [1994] and Robertson et al., “J. Am.Chem. Soc.” 113:2722-2729 [1991]), but ordinarily, the NP-encodingnucleic acid (generally DNA) is inserted into an appropriate expressionvector, host cells are transfected with the recombinant vector, therecombinant host cells are grown in suitable culture medium, and thedesired amino acid sequence NP is recovered from the recombinant cellculture by chromatographic or other purification methods. It is alsowithin the scope of this invention to partially synthesize the NP inrecombinant cell culture or by in vitro methods and then ligate thepolypeptide fragments by peptide ligase (reverse proteolytic synthesis).

[0086] The nucleic acid for expression of the NP typically encodes apreprothrombin containing the desired mutation since this permits facileexpression and processing by methods heretofore used with thrombin. Inaddition, known methods for the recombinant expression of“gla-domainless” prothrombin are readily adapted to the preparation ofthe NPs of this invention. It is within the scope of this invention toexpress nucleic acid that encodes NP analogues of any of (a)prethrombin-2 (alpha thrombin) or prethrombin-1, (b) the sequence ofboth chains of meizothrombin des 1 (which are either expressed as asingle chain extending from S156 [Degen et al.] to the 3′ end of thenucleic acid encoding the B-chain, or are coexpressed as nucleic acidsseparately encoding the S156 fragment and the thrombin B chain), (c)thrombin (which is expressed as a single chain encoding both of the Aand B chains, or is coexpressed in the same cell as nucleic acidsseparately encoding the A and B chains) or (d) the thrombin B chain freeof the A chain. By “separately encoding” is meant that the A and Bchain-encoding nucleic acids are separated by at least a stop codon and,if necessary, the downstream nucleic acid (usually B chain) commenceswith a start codon. Note, however, that bacterial polycistronicexpression cassettes may not require a start codon for the downstreamcoding sequence. Suitable vectors and host cells for expression of thetwo chains independently are described in U.S. Pat. No. 4,923,805. Suchheterodimeric expression systems generally are mammalian.

[0087] Polycistronic expression systems are useful for single cellcoexpression of sequences comprising the two thrombin chains describedabove. These systems are known per se, and are particularly usefulwhere, as here, it is desired to make the NP A and B chainsindependently in the cell culture and thereby avoid any need forpost-translational processing. In this instance both chains generallyare expressed under the direction of the same expression controlsequence (which chains and expression control sequences can be on thesame or a different vector). However, the nucleic acid encoding eachchain contains its own start codon as required and, preferably, eachnucleic acid encodes its own signal sequence.

[0088] The NP optionally is expressed as a preprotein of α-thrombin orthe A or B chains separately, whereby the NP is expressed as a precursorthat is processed to the mature NP and secreted from the host cell. Thepresequences or signal sequences for use herein include the nativepresequence normally associated with the source of the thrombin gene,e.g. the human presequence for preprothrombin, or presequences that haveamino acid sequences that are heterologous by sequence or origin to theprothrombin signal. Suitable presequences include those of (a) microbialproteases such as subtilisin, (b) mammalian proteases such as trypsin,chymotrypsin, fibrinolytic enzymes including urokinase or tPA, and bloodclotting factors such as Factors V, X, IX, VII, VIII or fibrinogen, (c)cytokines such as gamma interferon or an interleukin, (d) growth factorssuch as growth hormone or TGF-alpha, (e) polypeptides or proteins havingN-terminal mature sequences that are homologous to human thrombin A or Bchains, (f) immunoglobulins, (g) receptors, (h) other presequences ofsecreted or cell membrane bound proteins or (i) known presequences usedto direct the secretion of gla domainless prothrombin (pages 11, line30-page 12, line 21 of WO 93/13208). Signal sequences optionally arederived from or are homologous to the host cell, or at least thephylogenetic branch to which the host cell belongs. For example, oneordinarily would use a presequence of a yeast protein, such as matingfactor, in a yeast expression system, or of a bacterium, such as ST-IIor beta-lactamase, in bacterial cell culture systems. It would bedesirable to select signals from heterologous polypeptides that havemature N-terminal residue(s) that are the same as the mature thrombin Aor B chains, and to use those signals with the N-terminally-homologousthrombin chain. A wide variety of suitable signal sequences are knownand can be used in methods for the preparation of the NPs describedherein.

[0089] The nucleic acid constructs encoding the secreted NP generallywill encode the thrombin A or B chains fused at their N-termini to thenormal C-terminus of the same or different signal sequences. They arespliced into expression vectors under the control of expression controlsequences such as promoters, operators, enhancers and polyadenylationsequences as is generally known in the art. Constructing suitableexpression vectors for secreted or protoplasmically expressed NPs ofthis invention is a matter of routine for those skilled in the art, andwill be accomplished using the conventional tools of molecular biology,including nucleic acid synthesis in vitro, PCR, adapters, ligases,restriction enzymes, expression and shuttle plasmids, transfection aidsand the like, all of which are publicly (and for the most partcommercially) available.

[0090] Suitable promoters or enhancers, termination sequences and otherfunctionalities for use in the expression of NP in given recombinanthost cells are well known, as are suitable host cells for transfectionwith nucleic acid encoding the desired NP, some of which are mentionedabove while others are described in WO 93/13208 at page 12, line 21-page19, line 5, and EP 319,312 B1, page 16, lines 10-18 and Table IIthereof. It may be optimal to use host cells that are capable ofglycosylating the NPs, which typically include mammalian cells such asembryonic kidney 293 cells, COS cells, CHO, BHK-21 cells and the like.In addition, host cells are suitable that have been used heretofore toexpress proteolytic enzymes or zymogens in recombinant cell culture, orwhich are known to already express high levels of such enzymes orzymogens in non-recombinant culture. In the latter case, if theendogenous enzyme or zymogen is difficult to separate from the NP thenthe endogenous gene should be removed by homologous recombination or itsexpression suppressed by cotransfecting the host cell with nucleic acidencoding an anti-sense sequence that is complementary to the RNAencoding the undesired polypeptide. In this case the expression controlsequences (e.g., promoter, enhancers, etc.) used by the endogenoushighly expressed gene optimally are used to control the expression ofthe NP.

[0091] The host-vector system should be selected so as to yieldsubstantially homogeneous NP, thereby avoiding the need to purify asingle molecular species from other isoforms of the NP. Thus, if thecell is capable of glycosylation, essentially all of the NP moleculesshould be glycosylated. In addition, host cells optimally will beselected that are devoid (in the relevant cell compartment) ofproteolytic activity that is capable of intrachain cleavage of thethrombin A or B chains. Cells can be selected that contain no protease,e.g., in the periplasm, that will cleave after thrombin B R62, R123,R73, or K154, all of which are known to be sites of B chain degradation.For example, E. coli and other microbial strains are known that possesslittle or no extracellular or periplasmic proteolytic activity (otherthan signal peptidases). Such cells optimally would be used inexpression systems in which the A and B chains are expressed in the samehost cell fused to the same or different signal sequences. The A and Bchains are co-secreted into the periplasm or extracellular medium wherethey become disulfide bonded. The absence of deleterious proteases helpsto ensure that the product is not rendered microheterogenous as to chainlength by host-endogenous proteases acting on the product NP, but ofcourse independent A and B chain secretion is not dependent upon the useof such cells. In addition, or alternatively, the basic residues of theA or B chains that are sites for proteolytic cleavage are substitutedwith residues other than K or R. For example, K154 has been reported tobe one of the sites cleaved in the conversion of gamma thrombin (Colmanet al., “Hemostasis and Thrombosis”, p. 154 (1987)). Table 1 above showsthat K154 can be substituted with A without significant change inactivity. Thus, K154 can be substituted with another residue other thanR in order to confer resistance to proteolytic degradation (in additionto whatever other variations are desired). This also may extend the invivo life of the NP.

[0092] The recombinant cells are cultured under conventional conditionsand using conventional culture media heretofore employed with the cellsin question. These conditions and media are widely known. Freshlytransfected cells may only transiently express the NPs, but stabletransformants readily are obtained in accord with conventional practiceusing cotransformation with a selection gene such as DHFR or glutaminesynthetase and serial culture in the presence of a selection agent suchas methotrexate or methionine sulfoximine, respectively. Yields of NPscan differ substantially despite minor differences in the character ofsubstituents or insertions. In such cases, it is desirable to screen foran expression system that will yield a quantity of thrombin that is atleast about 75% of that obtained with the reference thrombin in the sameexpression system. It occasionally is useful to culture the cells atlower than the usual 37° C., typically about from 10° C. to 30° C.,optimally about from 15° C. to 27° C. This is the case with eithermicrobial or mammalian cells.

[0093] The NP preferably is expressed as a properly assembled, disulfidebonded thrombin A and B chain analogue, or as the B chain analoguealone. In general, the NP will be water soluble. It may be expressed inbacteria in the form of refractile bodies, in which case the insolubleNP is recovered and refolded using known methods, e.g. dissolution inguanidinium hydrochloride followed by gradual removal of the denaturant.Directly expressed NPs of this invention may have an extra N-terminalmethionine or blocked methionine residue, although host cells can beemployed that will cleave away such extraneous N-terminal methionineresidues.

[0094] If the A and B chains are fused, for example when the NP isexpressed as the α-thrombin analogue, then post-translationalproteolytic processing may be required in order to activate the precusorzymogen to the proteolytically active NP. Such precursors are analogousto naturally occurring prothrombins or may be fusions of one or both NPchains with a thrombin-heterologous polypeptide, as in the case ofsignal sequences. Proteolytic activation and/or processing isaccomplished in the host cell culture itself, or can be done afterrecovery of the NP precursor (with or without intervening purificationof the NP precursor). Post-translational proteolytic processing (eitherwithin the host cell culture or after initial recovery of the NPprecursor) is used to remove any prothrombin (orprothrombin-heterologous) sequences that may be fused N-terminal to theA or B chain NPs, or that is inserted elsewhere within an NP precursor(e.g., antigen tags used to facilitate purification). The NP precursorsare hydrolyzed by an enzyme or enzymes that is capable of making thecorrect cleavages without excessive or undesirable hydrolysis within theNP A or B chains. A generally suitable enzyme for removing pro sequenceand activating native prothrombin is found in Echis carinatus(saw-scaled viper) venom. Factor Xa also is useful to activate NPprecursors. Proteolytic activation is not required by NP mature B chainor coexpressed individual mature A and B chains.

[0095] Proteolytic activation of NP precursor is facilitated bysubstitution of the thrombin activation domain (the Factor Xa cleavagesite) with another sequence recognized and cleaved by a differentprotease or by thrombin itself. Suitable substituted activation sitesfor use with the NPs of this invention are described in WO93/13208, page9, line 21-page 10, line 17. As noted in WO93/13208, substitution of thethrombin activation site in prothrombin with the yeast KEX2 site isattractive because yeast can express KEX2 and therefore endogenouslycleave thrombin precursors at the activation site. Other sequences thatare suitably substituted for the thrombin activation domain aredisclosed in Table 1 of EP 319,312 B1. These are cleaved by cellmembrane-associated proteases as part of cell culture processing of NPsor their precusors.

[0096] Proteolytic activation can be accomplished at any point in theexpression or purification of NP or its precursors, but typically isdone after purification of NP precursors from the cells and/or cellculture supernatant. It will be noted that NPs containing a new dibasicsite resulting from an insertion or substitution of an R or K residueinto the thrombin sequence may be susceptible to hydrolysis by enzymesthat otherwise would not cleave native thrombin, thereby reducing yieldssomewhat during expression or activation. In this case, it may bedesirable to select an expression system and/or an activating enzymehaving different substrate specificity so as to reduce adventitiouscleavage.

[0097] The yields of NP from recombinant host cell culture will vary,depending upon a number of factors including the culture conditions andthe nature of the NP. Optimally, the yield of NP by weight from theculture should be greater than about half (and preferably greater than75%) that of reference thrombin.

[0098] It is possible to diagnostically use the NP-containing,activated, concentrated conditioned medium of the recombinant cellswithout further purification. However, NPs intended for therapeutic useshould be isolated or purified by methods heretofore employed forthrombin or other proteins, e.g., native or reducing/SDSelectrophoresis, isoelectric focusing, immobilized pH gradientelectrophoresis, salt precipitation, solvent fractionation (usingethanol for example) and chromatography such as gel filtration, ionexchange (cation or anion), ligand affinity (cibacron blue F3GA orp-aminobenzamidine), immunoaffinity, chromatofocusing, reverse phase orhydrophobic interaction chromatography. Suitable methods are disclosedin Colman et al., “Hemostasis and Thrombosis”, p. 148 (1987) andreferences cited therein, in WO 93/13208 page 19, line 33-page 21, line25, and other conventional sources. The activating enzyme (if any) canbe immobilized or is removed in subsequent purification steps.Typically, the NP will be isolated so as to be >95% pure by weight ofprotein, and preferably greater than 99% pure.

[0099] The NPs or their fragments also are prepared in vitro, especiallyif they are relatively small, e.g. on the order of about 30 residues orless. However, larger and intact NPs also are prepared by in vitroprocesses. For example, smaller NPs are prepared by synthesis usingstandard solid-phase peptide synthesis procedures as described byMerrifield “J. Am. Chem. Soc.” 85:2149 (1963). These then are ligatedtogether by the use of peptide ligase (reverse proteolysis). In vitromethods of protein synthesis also are used to prepare NPs without theneed for recombinant cell culture. Such methods are useful forsmall-scale preparations, and have the advantage of reducing thepossible effect on yields of host cell proteases. In vitro NP proteinsynthesis has one additional quite substantial advantage in that itpermits the site-specific introduction into the NP of a non-naturallyoccurring amino acid residue (Benner, and Robertson et al., citedabove). Accordingly, when the term “amino acid residue” is used herein(in connection with thrombin modification by substitution or insertion,especially by a single amino acid) it will be understood that the aminoacid is not limited to the naturally occurring residues associated withnative tRNAs. As noted by Robertson et al., aminoacyl tRNA isefficiently prepared using a variety of non-naturally occurring aminoacid residues (“non-naturally occurring” means not naturally found inproteins, although the amino acid might be found in biological systemsin nature). Since the tRNA is selected to be incorporated at a codon notrecognized by any of the common tRNAs involved in protein synthesis, theselected non-naturally occurring amino acid residue is incorporated onlyat the particular site in the NP sequence chosen for the unique codon.Thus, in these cases the NP is encoded by a nucleic acid having anonsense codon, e.g., UAG, at the desired unique insertion orsubstitution site. The non-naturally occurring amino acids that aresuitably incorporated are described for example in Greenstein et al.,“Chemistry of the Amino Acids” Vols. 1-3, 1986. In general, one will usepharmaceutically innocuous L-amino acids that are found in nature butordinarily not incorporated into proteins. Such amino acids typicallywill be structurally related to a naturally occurring residue thatproduces the desired effect at a given site and will be used to furtherresolve and optimize the desired property of the NP.

[0100] The NPs of this invention also include thrombins that have beensubstituted by a non-peptidyl moiety, either for purposes of preparingthe NP to begin with or as a subsequent modification of the NP preparedby amino acid substitution, insertion or deletion as described elsewhereherein. NPs per se are prepared by covalent modification of thrombin orits component chains or subfragments. Since we have determined a numberof the key residues involved in the procoagulant and anticoagulantfunction of thrombin it is within the scope of this invention tointroduce a covalent modification at such sites that accomplishesessentially the same objective as the corresponding site-directed mutantwith a naturally occurring residue. For example, carboxyl-containingside chains of residues such as E229 are derivatized by reaction withcarbodiimides (R′—N═C═N—R′, where R and R′ are different alkyl groups),or are amidated by reaction with ammonia or substituted amines.

[0101] Basic side chains such as those of R233 and K52 also arederivatized. For example, R233D and E are very good PCAs in which apositively charged side chain has been substituted with a negativelycharged side chain. Similar charge reversals are accomplished bysubstituting a thrombin R233 or K52 with chloroacetic acid. Lysineresidues are acetylated with acetic anhydride.

[0102] Tryptophan is a relatively rare amino acid in thrombin.Accordingly, W50 is an attractive site for post-translational covalentmodification because substitution at other sites is expected to be lessthan may be the case with more common residues. Reaction of W50 with anoxidant such as a halogen donor, e.g. bromine, will yield the side chainstructure

[0103] This reaction should be conducted in aqueous solvent and at lowhalogen concentrations.

[0104] Other covalent modifications of NPs, or of thrombin to obtain theNPs of this invention, will be apparent to the artisan. Side chainswhere reaction is undesired are protected by masking them withantibodies directed against an epitope that includes the residue to beprotected. Reagents for accomplishing such modifications are well-knownand have been widely used in the diagnostic and preparative fields. SeeT. Creighton, Proteins: Structure and Molecular Properties, 1983.

[0105] Covalent modifications also are useful to accomplish objectivesother than the preparation of NPs having segregated substratespecificity. For example, NPs are rendered insoluble by cross-linkingthem to a water insoluble matrix. This is accomplished by reacting theNP and matrix with a bifunctional agent that forms the covalentcrosslink. Examples of suitable agents are1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, NP is immobilized on reactive water-insolublematrices such as cyanogen bromide-activated carbohydrates and thereactive substrates described in U.S. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440.

[0106] NPs also are covalently modified by linking the NP to variousnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth for example in U.S.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0107] The in vivo circulating half-life of NP is lengthened byconjugating the NP to a polymer that confers extended half-life, such aspolyethylene glycol (PEG). PEG is a non-immunogenic, linear, unchargedpolymer with three water molecules per ethylene oxide unit. (Maxfield,et al., “Polymer” 16:505-509 (1975); Bailey, F. E. et al., in “NonionicSurfactants”, Schick, M. J., ed, pp. 794-821 (1967)). Severaltherapeutic enzymes have been conjugated to PEG to increase their invivo half-life (Abuchowski, A. et al., “J. Biol. Chem.” 252:3582-3586(1977); Abuchowski, A. et al., “Cancer Biochem. Biophys.” 7:175-186(1984). An IL-2 (interleukin-2)-PEG conjugate has been reported toincrease circulatory life and potency (Katre, N. V. et al., “Proc. Natl.Acad. Sci.” 84:1487-1491 (1987); Goodson, R. et al., “Bio/Technology”8:343-346 (1990)). See also Abuchowski, A. et al., “J. Biol. Chem.”252:3578-3581 (1977). Any of the methods for PEG conjugation used inthese citations is acceptable for use with the NPs of this invention.

[0108] Finally, NPs are covalently modified for use in diagnosticapplications by cross-linking them to detectable groups heretoforeemployed in diagnostic assays as is more fully described below.

Uses for the NPs of this Invention

[0109] The NPs of this invention are useful in therapeutic, diagnosticand preparatory methods. Their use will depend upon the properties thatthey possess, as will be apparent to the ordinary artisan. For the mostpart, all of the NPs will retain immune epitopes of thrombin, so theyare useful in place of thrombin in immunoassays for thrombin, whether ornot they fall within the definition of PCA or FCP used herein andwhether or not they possess any proteolytic activity. In addition, PCAsand FCPs have specialized therapeutic uses.

[0110] Anticoagulant NPs, in particular PCAs, are useful to ameliorateor prevent undesired clotting. They are effective in activating theendogenous protein C pathway, serving as a potent anticoagulant bygenerating endogenous aPC to inactivate FVa and FVIIIa, and enhancingtPA mediated fibrinolysis by inactivating PAI-1. The clinicalindications for such NPs, and particularly PCAs, include thromboticdiseases or conditions such as septic shock, adjunctive therapy incoronary thrombolysis and angioplasty, pulmonary embolism, transientischemic attacks and strokes, unstable angina, M.I., deep venousthrombosis and a variety of arterial and venous thromboses. In a methodfor treating septic shock the anticoagulant NP is administered topatients in advanced sepsis (gram negative, gram positive or fungal),e.g. who exhibit symptoms of high fever, hypotension, disseminatedintravascular coagulation, renal insufficiency and/or ARDS.Anticoagulant NPs especially PCAs are useful for any of the utilitiesheretofore proposed for aPC. In this regard, see EP 191 606 B1, page 13,line 52, page 15, line 32. Thus, for therapeutic applications,anticoagulant NPs and especially PCAs are administered to a mammal,preferably a human, in a pharmaceutically acceptable dosage form and ina therapeutically effective dosage. The anticoagulant NP is administeredintravenously as a bolus or by continuous infusion over a period oftime, or by intramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, topical, or inhalation routes. The anticoagulant NPs alsoare suitably administered by catheter to exert primarily localanticoagulation.

[0111] Anticoagulant NPs also are useful in the diagnosis of clottingdisorders. A substantial portion of hypercoagulation states found inpatients cannot be ascribed to a particular molecular defect. Manypatients prone to bouts of thrombosis may suffer from an inborn error ina component of the protein C activation pathway, but it is presentlydifficult to diagnose such patients. PCA especially are useful toprovide a diagnosis of a defective activated protein C pathway and thecomponent of the pathway that is deficient. In this embodiment thesubject does not need to be in immediate need of anticoagulant therapybut may be known to have been subject to excessive thrombosis of unknownorigin. Anticoagulant NP such as PCA is administered to the patient andthe anticoagulant state of the patient's blood is determined after thePCA has had time to act. The dose of PCA is substantially the same asthe amount of PCA effective in normal patients in inducing detectableanticoagulation. The anticoagulation state of the subject is measured atsubstantially the same time points as those at which detectableanticoagulation is induced in normal patients. Any assay for bloodclotting is suitable, e.g. aPTT and the like. If the PCA is notsuccessful in detectably anticoagulating the subject's blood it islikely that the patient suffers from a defect in thethrombomodulin-protein C anticoagulation pathway. In a furtherembodiment, administering soluble TM together with PCA and determiningwhether either protein resolves the defect will yield information on thelocation of the deficiency, i.e., if TM and the NP induceanticoagulation, but protein C and the NP do not, one can conclude thatthe subject's TM is responsible for the defect.

[0112] Procoagulant NPs are useful therapeutically as procoagulants forany purpose. For example, they are impregnated into dressings, bandagesand the like, or otherwise included in dosage forms intended for topicaladministration. FCP also are effective as thrombosis accelerants in thethrombotic treatment of large solid tumors. The FCP are used in place ofC4b binding protein, or anti-aPC antibodies as described in U.S. Pat.No. 5,147,638, optimally in combination with a cytokine such as TNF-α orTNF-β, gamma interferon, IL-1, IL-2 and/or GM-CSF. The dose of FCP willbe titered to induce clotting in tumors, but not produce clinicallysignificant clotting elsewhere. Proforms of NPs which are dependent uponendogenous activation also can be used in this method.

[0113] Dosage forms of the NPs of this invention are thoseconventionally used with other protein therapeutics. The NPS will besterile, typically will be disposed into containers suitable for sterileaccess (vials, for example, sealed with elastomeric stoppers), andencompass pharmaceutically acceptable carriers that are nontoxic,including salts and solvates. Examples of such carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins such ashuman serum albumin, buffers such as phosphates, glycine, arginine,lysine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, alkali metal salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and polyethylene glycol. Carriers for topical or gel-basedforms of the NPs include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol, and wood wax alcohols. Conventional depot forms maybe used and include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, and sublingualtablets. The NP typically will be formulated in such vehicles at aconcentration of about 1-150 micromolar or 1-200 mg/mL. It will bedesirable to include an antioxidant such as ascorbate, particularly if,as will generally be the case, the NP includes a disulfide bond joiningthe A and B chains or a disulfide bond found within the B chain. The pHof the formulation should be selected to minimize deamidation andautolysis, and generally will be about from 5 to 8. The formulationspreferably will be lyophilized, but can be prepared and stored asaqueous solutions.

[0114] The appropriate dosage of anticoagulant NP for the prevention ortreatment of thrombosis will depend on the amount of residualprocoagulant activity (if any), location and nature of the thrombosis orexpected thrombosis to be treated, the severity and course of theclotting disorder, whether the anticoagulant NP is administered forprophylaxis or therapy, the nature and course of previous treatment, thehalf-life of the NP in the circulation, the use of other therapeutics oranticoagulants, the locality of NP administration (local doses will belower than systemic doses), the patient's response to the NP, whetherthe NP requires activation by endogenous prothrombin activation pathways(i.e., whether the thrombin NP is supplied as the correspondingprothrombin or unactivated fragment thereof) and other considerationswithin the discretion of the attending physician. Since PCA is an activeenzyme, the amount required for anti-thrombotic effect is quite small,in the 5-10 nM range (final concentration in plasma), and in fact ourstudies in rabbits show that the K52A PCA produces therapeuticanticoagulation at plasma levels ranging from about 3-9 nM, with similarresults for E229A PCA. This compares quite favorably with otheranticoagulant macromolecules such as hirudin (100-800 nM) or GS-522, athrombin-binding DNA aptamer (2000-4000 nM; PCT US 92/01367). Ourpreliminary monkey studies suggest that the dose of PCA in primates willrange about from 0.05 U/kg/min to 20 U/kg/min, depending primarily onthe amount of residual fibrinogen cleavage activity (which will militatein favor of lower doses to prevent fibrinogen consumption and plateletactivation) and the potency of the aPC activity (PCA that retain asubstantial proportion of wild-type aPC activity will be used in thelower dose range). 1 U is equal to the amount of S-2238 amidolyticactivity in 1 NIH Unit (clotting activity) of wild-type thrombin (cfExample 4).

[0115] The NPs of this invention are not expected to be immunogenicsince in preferred embodiments only a few critical residues are modified(less than about 5), or are masked with glycosylation. This isparticularly the case with single substitutions (e.g. E229D) that arehighly conservative. The half-life of certain NPs may be very short,requiring them to be used as a continuous infusion (the plasma half-lifeof wild type thrombin in vivo is probably extremely short, in the rangeof seconds). This, however, may be clinically advantageous in somecircumstances since it would dispense with the need for a thrombininhibitor should a bleeding episode develop in the patient. PCAs have ananticoagulant effect in subhuman primates and other animals for aboutfrom 90 to 180 minutes following administration at adequate doses, so itis feasible to administer PCAs by a single injection or a brief (e.g.,10 minutes) infusion. However, administration by continuous infusion isalso within the scope of this invention.

[0116] Procoagulant or anticoagulant NPs suitably are administered tothe patient at one time or over a series of treatments. For repeatedadministrations over several days or longer, depending on the condition,the treatment is continued until the desired anticoagulant orprocoagulant effect is achieved.

[0117] According to another embodiment of the invention, theeffectiveness of the compounds of this invention as procoagulants oranticoagulants is improved by administering the compounds serially orsimultaneously with another agent or medical procedure that is effectivefor the relevant purposes. PCA is useful in conjunction with knownanticoagulants such as heparin, Factor X inhibitors, plateletaggregation inhibitors, and the like. It also is useful in conjunctionwith thrombolytic therapies using for example tPA, urokinase orstreptokinase, or with balloon angioplasty or other procedures thatentail the lysis of clots, treatment of atherosclerotic plaques ormanipulation of or contact with the vascular endothelium.

[0118] The NPs of this invention are useful in identifying substancesthat bind to target sites on thrombin (thrombin binding partners, or“TBP”). Of particular interest are substances that bind to the PCactivating domain of thrombin but not to the procoagulant domain, orvice-versa. In preferred embodiments they are capable of binding tothrombin and substantially inhibit the procoagulant activity of thrombinwithout comparatively inhibiting its PC activating function. This doesnot mean that the TBP need leave the thrombin PC activating functionentirely uninhibited. All that is necessary in this instance is that thedegree of inhibition of PC activation be less than that of theprocoagulant function. Typically, the TBP will inhibit the targetthrombin proteolytic activity such that the ratio of PC activation toprocoagulant activity is less than about 0.5 or greater than about 2.0.TBPs in general are polymers falling into two classes: polypeptides andoligonucleotides, but are essentially unlimited and can include anysubstance, including molecules of less than about 1500 molecular weight.TBPs are used in diagnostic procedures, to indirectly label thrombin orto separate thrombin from other substances present in test samples.TBP's ability to bind to thrombin also is useful in methods forrecovering thrombin from contaminated mixtures such as cell culturesupernatants of recombinant thrombin-expressing cells (including thethrombin amino acid sequence NPs herein). Typically, NPs can be used inplace of thrombin standards in immunoassays for thrombin if they possessat least one thrombin epitope recognized by the antibody used in thethrombin immunoassay in question, while the TBPs are used in place ofantibodies for thrombin. The NPs also are useful, as appropriate, infunctional assays for certain individual properties of thrombin, forexample its procoagulant activity provided that the NP retainsprocoagulant activity.

[0119] Polypeptide TBPs typically are peptides or proteins which arecapable of specifically binding FCP but are not capable of bindingsubstantially to PCA, and vice-versa. They thus are highly specificinhibitors of either the procoagulant or aPC function of thrombin.

[0120] Peptide TBPs are obtained by the use of in vitro directedevolutionary methods such as those employing filamentous phage topresent candidate sequences (otherwise known as phage display) andsimilar methods known per se which rely on the systematic generation andscreening of peptides for activity. These typically are rather smallmolecules, containing on the order of about 5 to 20 residues. The FCPand PCA polypeptides of this invention are useful in screening for suchpeptide TBPs in the same general fashion as they are in screening foroligonucleotide. TBPs.

[0121] Antibody TBPs are immunoglobulins, ordinarily monoclonalantibodies, which are capable of specifically inhibiting theprocoagulant or aPC function of thrombin. Antibodies are raised againsta protein composition comprising a reference sequence thrombin. In orderto obtain an anticoagulant antibody the antibody pool is adsorbed ontoan PCA, whereby antibodies that do not substantially bind (but which dodemonstrate clotting inhibition) are recovered. These antibodiesnecessarily will be directed to fibrinogen cleavage epitopes. Ananalogous strategy is used to prepare a procoagulant antibody.

[0122] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare essentially identical in specificity and affinity. Monoclonalantibodies include hybrid and recombinant antibodies (e.g. “humanized”antibodies) regardless of species of origin or immunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab′)₂,and Fv). Thus, “monoclonal” antibodies are produced by any particularmethod that will yield a substantially homogeneous population. Forexample, monoclonal antibodies may be made using the methods describedby Kohler & Milstein, “Nature” 256:495 (1975), Goding, MonoclonalAntibodies: Principles and Practice pp. 59-103 (1986), Kozbor, “J.Immunol.” 133:3001 (1984), or Brodeur, et. al., Monoclonal AntibodyProduction Techniques and Applications. pp. 51-63 (1987), or may be madeby recombinant DNA methods. Cabilly, et al., U.S. Pat. No. 4,816,567.

[0123] In a preferred embodiment of the invention, the monoclonalantibody will have an affinity for reference sequence thrombin of atleast about 10⁹ moles/liter, as determined, for example, by theScatchard analysis of Munson & Pollard, “Anal. Biochem.” 107:220 (1980).Also, the monoclonal antibody typically will inhibit the procoagulant oranticoagulant activity of thrombin at least about 50%, preferablygreater than 80%, and most preferably greater than 90%, as determined,for example, by the PC activation assay or thrombin time determinationsdisclosed herein.

[0124] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such assimian COS cells, Chinese Hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells.

[0125] The DNA optionally may be modified in order to change thecharacter of the immunoglobulin produced by its expression. For example,humanized forms of murine antibodies are produced by substituting acomplementarity determining region (CDR) of the murine antibody variabledomain for the corresponding region of a human antibody. In someembodiments, selected framework region (FR) amino acid residues of themurine antibody also are substituted for the corresponding amino acidresidues in the human antibody. Humanized forms of murine antibodiesalso may be produced by substituting the coding sequence for human heavyand light constant chain domains in place of the homologous murinesequences. Morrison, et al., “PNAS” 81:6851 (1984).

[0126] Evolutionary selection methods for oligonucleotides that bind totarget proteins are well known (WO 92/14843; Ellington et al., “Nature”355:850 (1992); Bock et al., “Nature” 355:564 (1992); Ellington et al.,“Nature” 346:818 (1990); Tuerk et al., “Science” 249:505 (1990). Theseoligonucleotides, commonly known as aptamers, generally contain theusual A, T, G, C or U bases or derivatives thereof, and comprisesequences that bind to a predetermined site on a target protein. In thiscase, the FCP and the PCA are used in negative (absence of binding) andpositive (binding) selection protocols to yield TBPs such as aptamersthat are specific for the inhibition of either clotting or aPC activity.A selection method for TBPs that inhibit the procoagulant function ofthrombin (but do not substantially interfere with its anticoagulantactivity) comprises (a) preparing a pool of candidates(oligonucleotides, peptides, extracts, proteins, etc.), (b) contactingthe candidates with thrombin having procoagulant activity (typicallyreference sequence thrombin) (c) isolating from the thrombin thosecandidates that are able to bind to thrombin, (d) contacting thecandidates from step c) with an NP in which the thrombin procoagulantfunction has been mutated substantially out of the NP, i.e., a PCA, and(e) recovering those candidates that do not bind to PCA. This ensuresthat any candidate so selected is capable of binding to the amino acidsite(s) that were varied in arriving at the PCA polypeptide, i.e., theresidues instrumental in thrombin's procoagulant function.Alternatively, a candidate or a pool enriched in potential anticoagulantTBP is selected by (a) preparing a pool of candidate binding partners(as above), (b) contacting the candidates with thrombin havingprocoagulant activity (typically reference sequence thrombin), (c)isolating from the thrombin those candidates that are able to bind tothrombin, (d) contacting the candidates from step c) with a thrombin NPin which the PC activating function has been mutated substantially outof the NP, i.e., a FCP, and (e) recovering those candidates that bind toFCP. The resulting candidate pool thus has been specifically enriched incandidates, or a candidate has been selected, that does not bind tothrombin at the mutated residues in the PC activation site. Thiscandidate pool is then selected for anticoagulant activity, e.g., byfurther selecting for TBPs that do not bind to PCA. In the end, thiswill enrich the pool or select for candidate TBPs that do not bind thePC activation site but which do bind to the procoagulant residues ofthrombin. Steps c) and d) optionally are reversed in either alternative.

[0127] TBPs that inhibit the protein C activating function of thrombinare selected by a process analogous to the enrichment of TBPs having theability to inhibit the procoagulant function as described above, e.g.,TBPs are selected that bind to thrombin but which do not bind to FCP.

[0128] The binding steps are accomplished in most cases usingimmobilized NP, typically NP that has been absorbed through its glycosylsubstituents or its N— or C-terminus to an insoluble carrier inaccordance with known methods (e.g., concanavalin A agaroseimmobilization followed by elution with alpha-methyl mannoside; Bock etal., op cit).

[0129] When the candidates are replicable moieties, e.g., filamentousdisplay phage or oligonucleotides, one may optionally interpose one ormore steps of amplification of the selected or isolated candidate poolbetween steps c) and d). Ordinarily, the candidates are not amplifiedbetween these steps. In addition, it is desirable to repeat steps a)-e)and, in most instances, to interpose an amplification step between stepse) and a). Amplification (usually accomplished by PCR in the case ofoligonucleotide candidates), is useful in enriching the pools capable ofdemonstrating the selected-for function.

[0130] FCP and PCA are particularly useful as intermediates in thepreparation of TBPs.

[0131] For diagnostic applications, TBPs or the NPs of this inventionoptionally are labeled with a detectable moiety. The detectable moietycan be any substituent which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²p, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; a radioactive isotopic label, such as, ¹²⁵I,³²p, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

[0132] Any method known in the art per se can be used to conjugate theTBP or NP to the detectable moiety. See the methods described supra andHunter, et al., “Nature” 144:945 (1962); David, et al., “Biochemistry”13:1014 (1974); Pain, et al., “J. Immunol. Meth.” 40:219 (1981); andNygren, J. “Histochem. and Cytochem.” 30:407 (1982). OligonucleotideTBPs are labeled in the conventional fashion heretofore employed in thediagnostic probe art.

[0133] The labeled NPs are employed in the detection for example ofproteins that bind to thrombin, e.g. antibodies. In addition, theunlabeled NPs are linked to anti-analyte antibodies (either covalentlyor by immunoadsorption) or to analyte-binding receptors and arethemselves used as labels by virtue of their ability to cleave labeledpeptides that are readily detected by fluorescent or colorimetricmethods. Typical receptors to which the unlabeled NPs are linked includecell surface macromolecules, e.g., LFA-1, VLA-4, mac-1, I-CAM1, I-CAM2,I-CAM3 or V-CAM1. Typical antibodies to which the unlabeled NPs arelinked include antibodies directed against proteins participating in theblood clotting cascade as well as endothelial cell antigens, tumorantigens or other cell surface macromolecules. Suitable methods forconjugating proteins or for labeling proteins are well known and areconveniently applied to conjugating or labeling NPs. For example, NPB-chain is fused at its N-terminus to the C-terminus of animmunoglobulin heavy chain or to the C-terminus of the extracellulardomain of a cell surface receptor.

[0134] The TBPs or NPs of the present invention optionally are employedin known immunoassay techniques, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays.Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (1987).Competitive binding assays rely on the ability of a labeled standard(which may be thrombin, or an immunologically reactive portion thereofsuch as a labeled NP of this invention) to compete with the test samplethrombin for binding with a limited amount of TBP. The amount ofthrombin in the test sample is inversely proportional to the amount ofstandard that becomes bound to the TBP. To facilitate determining theamount of standard that becomes bound, the TBP generally isinsolubilized before or after the competition, so that the standard andanalyte that are bound to the TBP conveniently are separated from thestandard and analyte which remain unbound.

[0135] Sandwich assays involve the use of two TBPs, each capable ofbinding to a different target portion, or epitope, of thrombin. In asandwich assay, the test sample analyte is bound by a first TBP which isimmobilized on a solid support, and thereafter a second TBP binds to theanalyte, thus forming an insoluble three part complex. David & Greene,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-TBP antibody that is labeled with a detectable moiety (indirectsandwich assay). For example, one type of sandwich assay is an ELISAassay, in which case the detectable moiety is an enzyme.

[0136] The TBPs and NPs of the invention also are useful for in vivoimaging, wherein a TBP or NP labeled with a detectable moiety isadministered to a host, preferably into the bloodstream, and thepresence and location of the labeled TBP or NP in the host is assayed.

[0137] The following exemplary material is offered by way ofillustration only and is not intended to limit the invention in anymanner. All patent and literature references cited herein are expresslyincorporated by reference.

EXAMPLE 1 1.1 Construction of Expression Vectors for the Production ofRecombinant Human Prothrombin

[0138] The entire 1869 base pairs of sequence encoding human prothrombinplus 12 base pairs of 5′ untranslated sequence and 97 bases of 3′untranslated sequence were isolated on a SmaI to XbaI fragment from acDNA clone of human prothrombin, BS(KS)-hFII (obtained from Ross T. A.MacGillivray, Dept of Biochemistry, Univ of British Columbia, Vancouver,Canada). This fragment was cloned into the eukaryotic expression vector,pRc/CMV (Invitrogen Corp, San Diego, Calif.). pRc/CMV was digested withHind III and the 3′ recessed ends were made blunt by filling in with theKlenow fragment of E. coli DNA polymerase I. The vector was furtherdigested with XbaI and ligated with the SmaI to XbaI fragment containingthe prothrombin coding sequence. The resulting construct, pRc/CMV-hPT,contains the prothrombin coding sequence flanked by sequences requiredfor high level transcription in mammalian cells: the promoter from theimmediate early gene of human cytomegalovirus and transcriptiontermination sequences and polyadenylation signals from the bovine growthhormone gene. This clone also contains the β-lactamase gene conferringampicillin resistance and the ColE1 origin of replication forpropagation and maintenance in E. coli. Also present is the origin ofreplication of filamentous bacteriophage, f1 for production ofsingle-stranded DNA in E. coli infected with the defective helper phage,M13KO7 (Vieira and Messing, 1987). This vector can be used forexpression of human prothrombin in transiently transfected mammaliancells, however the presence of the neomycin phosphotransferase genemeans that permanently transfected cell-lines for large scale expressioncan be selected by resistance to the antibiotic, G418.

1.2 Mutagenesis Strategy to Identify Functional Residues on the Surfaceof Thrombin

[0139] Our mutagenesis strategy was designed to systematically scan thesurface of human α-thrombin to identify residues important forfibrinogen clotting, thrombomodulin-dependent protein C activation andinhibition of clotting by heparin/antithrombin III and the thrombinaptamer. The strategy was designed to maximize the chances ofidentifying functional residues while minimizing the opportunities forthe non-specific disruption of protein conformation. The charged andpolar (R, K, D, E, H, S, T, Q, N, Y, W) amino acids were of particularinterest for mutation as these residues are capable of participating inhydrogen bonds and electrostatic interactions that are likely to beimportant for the binding of charged ligands. Secondly, only the chargedand polar residues that are highly exposed to solvent on the surface ofα-thrombin were selected for mutation. The solvent accessible surfaceresidues were targeted as these residues are available for interactionswith ligands and are more tolerant of sequence variation (Bowie et al.,1990). The fractional accessibility (Rose et al., 1985) to a solventprobe of radius 1.4 Å was determined for each residue in thrombin in the2.3 Å crystal structure of human α-thrombin complexed with the inhibitorhirudin (Rydel et al., 1990) and the 70 charged and polar residues witha fractional accessibility of>³⁵% were selected for mutation. Only asingle residue, R36a, was excluded from this list. R36a is located atthe junction between the A and B chains of α-thrombin and is requiredfor the processing of prothrombin to mature, two chain, α-thrombin.However, several residues were added to the list, including fiveresidues (R20, K65, H66, R68, K77) located in the presumedfibrinogen-binding exosite and two residues (R98, W249) in the putativeheparin-binding site. Thus, a total of 76 residues were replaced withalanine by oligonucleotide-directed mutagenesis. Alanine was used forall substitutions because alanine is compatible with both α andβ-secondary structures (Klapper, 1977), tolerated in both buried andexposed locations in proteins (Klapper, 1977; Rose et al., 1985) and thenonpolarity and small size of its side chain ensures that substitutionwith alanine is less likely to disrupt protein conformation. Multiplesubstitutions were made simultaneously when two or three targetedresidues were clustered together. If such multiple mutants displayed afunctional phenotype then the individual residues were subsequentlysubstituted individually. The complete list of alanine replacementmutants is included in Table 1. The numbering system for residues inhuman α-thrombin is that used previously (Wu et al., 1991), where theresidues in the B-chain are numbered consecutively (1-259). The residuesin the A-chain are also numbered consecutively (1a-36a) but are suffixedwith lower case ‘a’ to indicate identity with the A-chain.

1.3 Construction of Alanine Substitutions in Human Prothrombin byOligonucleotide-directed Mutagenesis

[0140] Alanine substitutions were introduced into the human prothrombingene by oligonucleotide-directed mutagenesis on a single-stranded DNAtemplate (Zoller and Smith, 1982). A uracil-containing single-strandedDNA template was generated in a dut−ung− strain of E. Coli (CJ236)(Kunkel et al., 1987) allowing selection against the non-mutant templatestrand in ung+ strains of E. coli following extension and ligation ofthe mutagenic oligonucleotide primer. Synthetic oligonucleotide primersencoding alanine substitutions flanked by regions of 12 nucleotidescomplementary to the prothrombin coding strand were synthesized on anApplied Biosystems Inc. solid phase synthesizer using phosphoamiditechemistry. Phosphorylated primers were hybridized with the pRc/CMV-hPTtemplate, extended by T7 DNA polymerase and ligated to the newlysynthesized strand by T4 DNA ligase. The heteroduplex DNA was used totransform dut⁺ung⁺ E. coli strain, XL1-Blue, to select against theuracil-containing parental strand. Single-stranded DNA from individualtransformants was sequenced using dideoxy-chain-termination andSequenase 2.0 (United States Biochemical) to confirm the identity ofeach mutation. 500 ml cultures of each pRc/CMV-hPT mutant in XL1-Bluewere used to produce plasmid DNA for transfection of cultured COS-7cells. Approximately 1 mg of closed circular plasmid DNA was isolatedusing the QIAGEN Maxi plasmid preparation kit. DNA encoding the otherNPs described herein is made in the same fashion using an appropriatetemplate.

EXAMPLE 2 2.1 Expression and Activation of Recombinant Prothrombins forTransient Expression Assays

[0141] Recombinant prothrombin constructs (10-20 μg) containing theunmodified prothrombin cDNA (for wild-type thrombin), thrombin mutatedat S205A (for a negative control), and the various mutants wereseparately introduced into 10⁶ COS-7 cells grown in a 35 mm well, by theDEAE-dextran method of transfection (Adams and Rose, 1985). Two dayspost-transfection, the cell monolayer was washed twice with PBS and 1 mlof serum free DME medium was added back to the monolayer and incubatedat 37° C. for 24 h (occasionally it was advantageous to culture attemperatures below about 30° C., in particular 27° C., when expressionis not detected or is poor at 37° C., i.e. Mt 11, 12, 13, 34, 35, 14.5and 37c). The conditioned medium was then harvested, centrifuged toremove any cell debris and concentrated 20-fold by ultrafiltration withCentricon-30. 50 μl of this concentrated medium were activated with 1.5μg of Echis carinatus venom at 37° C. for 45 min. 12.5 μl ofconcentrated conditioned medium before and after venom activation wereanalyzed by Western blotting using monoclonal antibody (labeled withalkaline phosphatase) directed against human thrombin. The protein levelwas estimated by comparing the intensity of the band with a seriallydiluted plasma thrombin standard. The expression level of the thrombinsvaries from 0.12 to 2.0 μg of thrombin per 10⁶ cells, as estimated byboth Western analysis and amidolytic assay.

2.1a Quantitation of Recombinant Prothrombins in Conditioned CellCulture Medium by Slot Blot

[0142] Thrombin protein concentration was determined by quantitativeWestern blotting using a Schleicher and Schuell Minifold II vacuumslot-blot apparatus. Prothrombin in 20-fold concentrated conditionedmedium and purified prothrombin standards (American Diagnostica) wasactivated as described above. Samples and standards were diluted withPBS and adjusted to the same concentration of conditioned medium frommock transfected cells. Duplicate 100 μl aliquots containingapproximately 50 ng activated prothrombin and duplicate aliquots ofpurified, activated prothrombin standards (1-200 ng) were aspiratedthrough a 0.45 μm nitrocellulose filter in the slot-blot apparatus. Eachslot was washed twice with 200 μl aliquots of PBS. The filter was washedtwice with PBS and blocked with 5% non-fat skim milk (Carnation) in PBS.The blot was incubated with 11 μg/ml rabbit polyclonal immunoglobulinsagainst human prothrombin (Dako) in 5% non-fat skim milk in PBS. Theblot was washed with PBS containing 0.05% Tween-20 and incubated with 1μCi/ml ³⁵S-labeled donkey F(ab′)₂ directed against rabbitimmunoglobulins (Amersham). The blot was washed with PBS containing0.05% Tween-20 and the radioactivity at each position was determined byscanning using an Ambis 4000 radioanalytic imaging detector. Thethrombin concentration in each sample was determined from the standardcurve which was linear over the range 1-200 ng prothrombin.

2.2 Amidolytic Assay

[0143] The hydrolysis by thrombin of the chromogenic substrate S-2238was performed as previously described (Wu et al., 1991). A standardcurve was constructed with plasma thrombin where 1 μg of thrombin givesa rate of hydrolysis of 1220.5 mOD/min in 300 μl of 100 μM S-2238. 20 μlof a one third dilution of venom activated conditioned medium were usedfor the measurement of the rate of hydrolysis of 300 μl of 100 μMS-2238.

2.3 Fibrinogen Clotting

[0144] The amount of venom activated conditioned medium that gives 335mOD/min (equivalent to 0.27 μg of plasma thrombin) in the amidolyticassay of Example 2.2 was used in fibrinogen clotting. The reactionmixture contained 20 μl conditioned medium and 180 82 l selection buffercontaining 20 mM Tris acetate pH 7.5, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂,1 mM CaCl₂. Reaction was initiated by addition of 50 μl of humanfibrinogen at 2 mg/ml freshly diluted in selection buffer from a stockof 10 mg/ml made in calcium free PBS. Time in seconds from addition offibrinogen to clot formation was measured with a fibrometer. A plasmathrombin standard clotting curve was used to convert the clotting timesinto mg/ml equivalent of plasma thrombin. Results are expressed as % ofwild type activity.

2.4 Protein C Activation

[0145] Cell lysates were prepared from TMnc cell expressing recombinanthuman thrombomodulin at the level of 504±34 fmoles/10⁶ cells (Tsiang etal., 1992) as previously described (Tsiang et al., 1990). About 8×10⁶cells were lysed in 800 μl, giving a thrombomodulin concentration of ˜5nM in the lysate. Control lysates were similarly prepared from theuntransfected parent CV-1 cell line which does not express TM.Commercially available human plasma protein C contains for each pmole ofprotein C ˜0.005-0.02 pmoles of contaminating prothrombin. To circumventthis problem, 444 pmoles of protein C were first treated with 10 μg ofEchis carinatus venom for 30 min at 37° C. to convert the contaminatingprothrombin into thrombin which was then inactivated by titration withthe thrombin inhibitor PPACK (D-Phe-Pro-Arg-Chloromethyl Ketone). Thisvenom-processed and PPACK-titrated protein C was then used in theprotein C activation assay (Tsiang et al., 1990). The assay mixturecontained an amount of venom-activated conditioned medium correspondingto a standard amount of S-2238 amidolytic activity (8.5 mOD/min), 20 μlof TMnc cell lysate and 887 nM protein C in a total volume of 50 μl.This mixture was incubated at 37° C. for 1 h and stopped by addition ofantithrombin III and heparin. For thrombomodulin-independent protein Cactivation, TMnc lysate was omitted and 2 mM CaCl₂ was replaced with 5mM Na₂EDTA in the assay mixture. The activated protein C generated wasassayed by hydrolysis of chromogenic substrate S-2366. The raw scores ofTable 1a reflect correction in the same fashion as in Example 2.3. Theprotein C activation activity was expressed as % of wild type activity.

2.5 Heparin-Dependent Antithrombin III Inhibition of Clotting

[0146] An amount of venom activated medium equivalent to a rate ofS-2238 hydrolysis of 370 mOD/min was used in each assay. The volume ofthe sample was adjusted to 15 μl with mock transfected 20-foldconcentrated medium, mixed with 135 μl selection buffer and pre-warmedat 37° C. Clotting time was measured immediately after simultaneousaddition of 50 μl of 2 mg/ml fibrinogen and 50 μl of 650 nM AT-III withor without 0.1 U/ml heparin, to the sample mixture. Sensitivity ofAT-III inhibition of clotting to heparin was expressed in % residualclotting activity in the presence of heparin relative to no heparincontrol.

EXAMPLE 3 3.1 Stable Expression of Recombinant Thrombins

[0147] Linearized recombinant prothrombin constructs from Example 1 (10μg) were introduced into BHK-21 cells using the calcium phosphate methodof transfection (Graham and Van der Eb, 1973; Parker and Stark, 1979).Clones were selected in culture medium containing the antibiotic, G418and the expression levels were determined by amidolytic activity andWestern blotting.

3.2 Production of Conditioned Medium for Protein Purification

[0148] Each desired BHK-21 clone expressing recombinant prothrombin wasseeded into 850 cm² roller bottles and grown in complete DMEM containing10% FCS (5×10⁶ cell per 200 mL per roller bottle). Two days later, afterthe cells reached confluency, microcarrier beads, Cytodex 2 (PharmaciaLKB, Piscataway, N.J.) were added to coat the cell monolayer. Three dayslater, after the beads were covered by cells growing on them, the cellswere washed with PBS and serum free DMEM containing 5 μg/mL insulin, 5μg/mL transferrin, 5 μg/mL fetuin and 10 μg/mL vitamin K was added backto the cells for prothrombin secretion. Conditioned medium was harvestedonce 3 days later and once more 6 days later.

[0149] In an alternative procedure, instead of using microcarrier beadsto expand the growth surface area, each desired BHK-21 clone was seededinto 1700 cm² expanded surface roller bottles in the same culture mediumas described above (5×10⁶ cells per 200 mL per roller bottle). Four dayslater, after the cells reached confluency, the cells were washed twicewith PBS (instead of 3 times) and the same serum free medium (150 mL)was added back to the cells for prothrombin expression. Conditionedmedium was harvested 3 times, once 4 days (150 mL) later, a second time8 days later (150 mL) and a third time 11 days later (100 mL).Conditioned medium was filtered through a Whatman N° 1 paper using aBuckner funnel to remove cell debris and, in the first method, detachedbeads.

3.3 Medium Concentration and Dialysis

[0150] Conditioned serum-free medium containing prothrombin wasconcentrated with a tangential flow filtration system (Pellicon^(R),Millipore, Bedford, Mass.). The low protein binding cellulose membraneof the tangential flow filter (type PLGC, 5 square feet, MWCO. 10 000)was pre-conditioned following the manufacturer's instruction. Duringconcentration, a pressure of 20-25 psi was used on the feeding-side, anda pressure of 3-4 psi was used on the retentate-side. The permeate flowrate was 150-200 mL/min. When the medium (retentate) was concentrated to˜500-600 mL, it was dialyzed 7-8 times against 1000-1200 mL of dialysisbuffer (0.1 M potassium phosphate, pH 7.5) through the same filter.Briefly, the dialysis buffer was added to the medium concentrate withgentle mixing by circulating the mixture in the filtration system forabout 2-3 min (permeation off). The mixture was then concentrated to500-600 mL. After repeating the above dialysis 7-8 times, greater then99.9% of the medium was replaced by the dialysis buffer.

3.4 Prothrombin Purification

[0151] The final dialysate (600-800 mL) was then filtered through a 0.45μm sterile disposable filter (Nalgene, Rochester, N.Y.) and loaded ontoa (2.6×7)cm DEAE-sepharose fast flow anion-exchange column (Pharmacia,Piscataway, N.J.). The prothrombin peak was eluted between 0.35-0.45 Mpotassium phosphate using a 0.1-0.7 M gradient. Aliquots of fractionscontaining prothrombin were determined by both amidolytic assay aftersoluble Echis carinatus venom activation (Sigma, St. Louis, Mo.) andSDS-PAGE. The active fractions were pooled and dialyzed against 0.02 MHEPES, 0.1 M NaCl, pH 8.0 at 4° C. over night. After dialysis the pooledprothrombin fractions were concentrated to 10-15 ml through a PM30membrane using a stirred cell (Amicon, Beverly, Mass.).

[0152] 3.5 Purification of NPs

[0153] Prothrombin NP in the concentrate was processed to NP by Echiscarinatus venom that was pre-adsorbed on Amberlite CG50 (ICNBiomedicals, Irvine, Calif.) and optionally immobilized to Affi-Gel 10beads (Bio Rad, Richmond, Calif.). Venom activation of the prothrombinNP was done with gentle rotation for 50 min at 37° C. The venom-beadswere removed by centrifugation followed by filtration through a 0.45 μmfilter. The filtrate was immediately loaded onto a (2.6×7) cm AmberliteCG50 (200-400 mesh) cation-exchange column. NP was eluted at 0.4 M as asingle peak using a 0.1-1.0 M NaCl gradient. NP fractions were pooledbased on amidolytic activity and SDS-PAGE (stained gel and Westernblot). Pooled NP fractions were then concentrated to 8-10 mL by using astirred cell with a PM30 membrane, and dialyzed against 0.1M NaCl, 0.02M HEPES, pH 8.0. The purified NP was characterized with respect toamidolytic activity plasma clotting time, protein C activation, andplatelet aggregation. Its purity and specific activity was alsodetermined. Finally the NP preparation was stored at −80° C. in aliquotsof 0.5 mL in sterile polypropylene tube.

3.6 Anticoagulation In Vivo Using NP

[0154] The formulations tested are specified below. Formulation ArticleDescription A control Sterile, isotonic saline, USP. B test A sterile,isotonic aqueous solution of wild-type human thrombin (HematologicTechnologies human thrombin from human plasma, prothrombinase activated)containing approximately 0.25 mg/mL thrombin. C control Recombinantreference sequence pro- thrombin activated with Echis venom in the samefashion as the test NP (FIG. 2A). D test A sterile, isotonic aqueoussolution of recombinant NP K52A containing approximately 0.25 mg/mL NP(FIG. 2B) E control A sterile, isotonic solution of rabbit brainthromboplastin (containing tissue factor).

[0155] The formulations were each administered by intravenousadministration to N.Z. white rabbits via an in-dwelling cannula placedin a central vein at a rate of infusion of about 13 mL/hour (14.3U/kg/min for wild-type and 11.7 U/kg/min for K52A). Because of thepotential thrombotic nature of the test formulations, it was thoughtthat infusion in a peripheral vein (e.g., the marginal ear vein) mightcause local thrombosis and ischemia due to a high local concentration ofthe test article. By accessing the central circulation (e.g., the SVCvia the jugular vein) this complication would be minimized due to thehigh intravascular flow rates and rapid distribution of an infusedcompound. This approach has been previously used in baboons using humanthrombin (J. Clin. Invest., 92:2003-12, 1993).

[0156] Infusion of formulation E was via a marginal ear vein as thisroute of administration has been shown to be safe and effective.

[0157] The results are shown in FIGS. 2A, 2B and 3. Each figurerepresents the results with one rabbit. FIG. 2A shows that wild-typethrombin causes substantial fibrinogen consumption and excessiveanticoagulation. In contrast, FIG. 2B demonstrates that K52A PCA iscapable of anticoagulant activity in the normal range (the shaded areain the Figures), that it possesses clinically useful persistance ofaction, and that it does not cause fibrinogen consumption. FIG. 3depicts elimination half-life and reversibility.

3.7 Protein C Activation by PCAs in Presence or Absence of TM

[0158] The protein C activation assay is described in Example 2.4.Unlike Example 2.4, there is no need to pretreat the protein C stockwith venom and PPACK because there is no venom in purified recombinantthrombin to activate the contaminant prothrombin in the protein C stock.Thrombin, thrombomodulin and protein C were used at concentrationsindicated in FIG. 6. The reaction was incubated for 1 h at 37° C. andassayed for aPC activity using chromogenic substrate S-2366. Thisexperiment demonstrates that the PCAs K52A and E229A remainthrombomodulin dependent.

EXAMPLE 4 Demonstration of Reversible Anticoagulation in CynomolgusMonkeys Using Protein C Activator 2 (PCA 2) (E229A) and K52A PCA

[0159] PCA 2 prepared as described in Example 3.4 and 3.5 was formulatedin sterile isotonic aqueous solution in a total volume of 10 mL. The PCA2 solution was intravenously infused continuously for 10 min into aperipheral vein of adult male cynomolgus monkeys at a rate of 60 mL/hrin a total volume of 10 mL. The infusion rate was controlled by use ofan infusion pump. Two concentrations were administered: (1) 1.5μg/kg/min (2 U/kg/min) and (2) 4.5 μg/kg/min (6 U/kg/min). (1 U isdefined in text above).

[0160] Blood samples were collected by puncture of a peripheral vein atthe following time points: immediately prior to the start of infusiont=0 and at intervals following the start of infusion t=5, 10, 20, 30,45, 60, 90, 120, 150 min. Samples were approximately 2 mL in volume andcollected into citrate. Plasma was obtained from whole blood within 10minutes. Anticoagulation was monitored by measuring the aPTT in afibrometer. Fibrinogen levels were determined from clotting assays usingfibrinogen deficient plasma.

[0161]FIG. 4 illustrates the results of infusing individual monkeys withthe two doses of PCA 2. Both doses resulted in prolongation of theclotting time beyond the anticipated therapeutic range (shaded region)without consumption of fibrinogen indicating that PCA 2 was devoid ofdetectable procoagulant activity in vivo. The effect was reversible,with the aPTT returning to normal approximately 170 minutes after theinfusion was stopped. The reversibility of the effect suggested thatcoagulation factors were not consumed during the infusion, anotherindication that PCA 2 had no detectable procoagulant activity in vivo.Analysis of the kinetics of the reversal suggests a half-life forclearance of approximately 50 minutes. This degree of persistence isbelieved to be clinically useful.

[0162] In a separate study, 2 U/kg/min of K52A PCA was prepared andinfused over 10 minutes as described above and compared with a 2U/kg/min E229A PCA in the above Example. The results are shown in FIG.5. Although fibrinogen levels with K52A PCA at 2 U/kg/min were notchanged, K52A PCA infused under substantially the same conditions, butat an overdose of 12 U/kg/min, resulted in an aPTT>10× control and afall in fibrinogen to<10%. 12 U/kg/min was an overdose for monkeysdespite the fact that it was well tolerated by rabbits at this dose(supra). Thus, doses in monkeys will generally be lower than in rabbits.

[0163] Platelet consumption and bleeding times were assayed and theresults are shown in Table 2. TABLE 2 E229A E229A K52A (2 U/kg/min) (6U/kg/min) (2 U/kg/min) Time (post-infusion)(min) 0 60 150 0 60 180 0 120Platelets 345 352 400 341 458 471 Bleeding Time 2 2 2 2 (min)

[0164] This data demonstrates that platelet function was preserved andbleeding times unchanged, in contrast to the results seen withgpIIb/IIIa inhibitors or thrombin inhibitors.

EXAMPLE 5 5.1 Expression and Use of B-chain NP Alone

[0165] In view of our observations that substitutions in the A chain ofthrombin had negligible effect on S-2238 hydrolysis, FC or PA it will beuseful to use the PCA B-chain alone as an anticoagulant or the FCPB-chain alone as a procoagulant. FCP or PCA B-chain is obtained from thetwo chain parent by reduction of the linking disulfide bond andrefolding (Hageman et al, Arch. Biochem. Biophys. 171:327-336 [1975]).Preferably, however, the B-chain is obtained by expression of nucleicacid encoding the NP B-chain alone. An expression vector for B-chain PCAor FCP is derived from vectors constructed for the expression ofprothrombin, prethrombin 1, prethrombin 2 (single chain α-thrombin) bydeletion of the intervening sequence between the codon encoding thecarboxyl terminus of the signal peptide and the amino terminal residueof the thrombin B chain (I1). Deletions are achieved byoligonucleotide-directed mutagenesis using chemically synthesizedoligonucleotides that encode the deletion as primers on asingle-stranded DNA template as described for construction of the alasubstitution NPs. Optionally, the codon for C44 is mutagenized to encodeA or S.

5.2 Expression of Prethrombin-1 and Prethrombin-2 as GST Fusion Proteinsin E. coli

[0166] Prethrombin-1 (amino terminal residue S156, Degen et al. residuenumbers) and prethrombin-2 (amino terminal residue T272, Degen et al.residue numbers) were expressed as soluble fusion proteins withglutathione S-transferase (GST) in the cytoplasmic compartment of E.coli. The expression vector used was pGEX-5X-1 (Pharmacia LKBBiotechnology) which contains the pBR322 origin of replication forpropagation in E. coli, the β-lactamase gene encoding for resistance tothe antibiotic ampicillin for maintaining the presence of the plasmidand the lac I^(q) gene encoding the lac repressor protein for dampeningbasal expression of the fusion protein. Expression is mediated by thetac promoter, which is inducible by IPTG, proximal to the ribosomebinding site and translation start codon of the GST gene. The sequenceencoding the 221 amino acids of GST is followed by a sequence (IEGR)encoding a proteolytic cleavage site for Factor Xa or Echis carinatusvenom that can be used to cleave the GST moiety from the fusion protein,and a polylinker sequence containing unique restriction enzyme sites forthe insertion of coding sequences to be fused to the GST codingsequence.

[0167] For Prethrombin-1, PCR primers were designed to amplify thenucleotide sequence encoding prethrombin-1 from S156 (Degen et al.residue numbers) to the carboxyl terminus of the thrombin B chain. The5′ end of the prethrombin-1 coding sequence was modified by the additionof a sequence encoding six consecutive histidine residues that can beused as an affinity tag for purification of fusion proteins onNi²⁺chelation matrices and the addition of a sequence encoding an EcoRIrestriction endonuclease site. The 3′ primer was modified to insert asequence encoding an XhoI restriction endonuclease site on the 3′ sideof the stop codon. For prethrombin-2, the same 3′ PCR primer was usedhowever the 5′ PCR primer was designed to fuse the prethrombin-2sequence starting at residue T272 (Degen et al. residue numbers) to thesix histidine affinity tag and EcoRI restriction site. PCR primers wereused to amplify the modified sequences encoding prethrombin-1 andprethrombin-2 using the prothrombin cDNA clone, BS(KS)-hFII as atemplate. The amplified fragments were cloned into the EcoRI and XhoIsites in pGEX-5X-1 in frame with the GST coding sequence. The resultingconstructs were used to transform E. coli strain JM 105 (ATCC 47016).

[0168] Stationary phase cultures of JM 105 containing QST-prethrombin-1and GST-prethrombin-2 were grown overnight at 37° C. in 25 mlLuria-Bertani medium (LB) containing 200 μg/ml ampicillin with shakingat 250 rpm. 200 ml of medium was inoculated with 4 ml of stationaryphase culture and incubated at 37° C. with shaking at 250 rpm untilcells reached exponential phase growth (O.D. at 600 run=0.6-1.2). Theincubation temperature was lowered to 17° C. and after 1 hour, IPTG wasadded to a final concentration of 1 mM and the incubation was continuedfor 24 hours. Bacterial cells were harvested by centrifugation at 5000g, washed with 50 mM TrisCl, 150 mM NaCl, pH 7.5. Cels were suspended in20 ml 50 mM TrisCl, 150 mM NaCl, pH 7.5, disrupted by sonication for 3-4minutes (50% duty cycle), Triton X-100 was added to a finalconcentration of 1% and the extract was mixed at 80 rpm for 60 minutesat 4° C. Insoluble material was removed by centrifugation at 10,000 g.GST-prethrombin-1 and GST-prethrombin-2 were expressed in the solublefraction at a yield of approximately 5 mg/liter of culture as assessedby Western blotting using a monoclonal antibody (EST-1) directed againsthuman α-thrombin. GST-prethrombin-1 and GST-prethrombin-2 fusionproteins were processed to mature α-thrombin by incubation with 30 μg/mlEchis carinatus venom in 50 mM TrisCl, 150 mM NaCl, pH 7.5 at 37° C. for30 minutes or longer. Processing of the fusion proteins to fragments ofthe same size as the A- and B-chains of human α-thrombin was visualizedby Western blotting following SDS-PAGE under reducing conditions. Uponprocessing, amidolytic activity towards the thrombin-specificchromogenic peptidyl substrate (S-2238) was assessed by incubating 200ng of processed GST-prethrombin-1 or GST-prethrombin-2 fusion proteinswith 100 μM S-2238 in 50 mM TrisCl, 150 mM NaCl, pH 7.5 at 37° C. for 10minutes. Amidolytic activity was only detected in samples afterprocessing with Echis carinatus venom.

[0169] GST-prethrombin-1 and GST-prethrombin-2 fusion proteins werepurified by affinity chromatography on glutathione-sepharose 4B(Pharmacia). Bacterial extracts were applied to a 1 ml column ofglutathione-sepharose 4B, the column was sealed and mixed for 40minutes. The column was drained and washed with 50 mM TrisCl, 150mMNaCl, pH 7.5 plus 1% Triton X-100, washed with 50 mM TrisCl, 150mM NaCl,pH 7.5 and eluted with 1 ml aliquots of 50 mM TrisCl, pH 8.0 containing10 mM glutathione. GST-prethrombin-1 and GST-prethrombin-2 fusionproteins eluted by 10 mM glutathione were approximately 50% pure asassessed by SDS-PAGE stained with coomassie blue.

[0170] The NPs of this invention are produced in the same fashion as theprethrombin-1 or prethrombin-2 in this example, and are processed tomature, activated NP as described herein, except that the constructs aremutagenized to introduce the desired sequence change into the expressionvector prior to expression in JM 105.

EXAMPLE 6 Platelet Aggregation by PCA 2 (E229A) and Wild-type Thrombin

[0171] Although PCA 2 was demonstrated to be defective in fibrinogenclotting, another important procoagulant function of thrombin is thestimulation of platelet aggregation as a result of cleavage of atransmembrane receptor on the platelet surface. In order to demonstratethat PCA 2 is also defective in this procoagulant function of thrombin,platelet aggregation studies were performed comparing differentconcentrations of PCA 2 and wild-type thrombin.

[0172] Platelet aggregation studies comparing wild-type and PCA-2thrombin were performed with citrated human platelet-rich plasma (PRP)using a Chrono-Log dual channel aggregometer (model 560-VS, Chrono-LogCorp, Havertown Pa.). Fresh human whole blood (4.5 mL) was collected insterile Vacutainer tubes (Becton Dickenson, Rutherford, N.J.) containing0.5 mL of 129 mM sodium citrate. PRP was separated from the citratedblood by centrifugation. The aggregometer was set according to themanufacturer's instructions. Recombinant human α-thrombin was added topre-warmed (37° C. for 2 min) PRP at final concentrations ranging from0.12 nM −0.49 nM for wild type and 0.74 nM −71.89 nM for PCA 2. Theplatelet aggregation stimulated by the thrombin addition to PRP wasmonitored by the increase in light transmission recorded on a chartrecorder for up to 5 minutes. The extent of platelet aggregation wasquantitated by measuring the area under the tracing 1.5 min after theaddition of thrombin. The extent of platelet aggregation (cm²) wasplotted against thrombin concentration (FIG. 7).

[0173] The EC50 (concentration required for half-maximal stimulation)for stimulation of platelet aggregation by wild type thrombin and PCA 2was estimated from the plot. PCA 2 (EC50=30.8 nM) is 8-times lesseffective than wild-type thrombin (EC50=3.9 nM) in stimulating plateletaggregation and thus is defective in this procoagulant activity as wellas in clotting fibrinogen.

1 2 1 885 DNA Homo sapiens CDS (1)..(885) 1 acc ttt ggc tcg gga gag gcagac tgt ggg ctg cga cct ctg ttc gag 48 Thr Phe Gly Ser Gly Glu Ala AspCys Gly Leu Arg Pro Leu Phe Glu 1 5 10 15 aag aag tcg ctg gag gac aaaacc gaa aga gag ctc ctg gaa tcc tac 96 Lys Lys Ser Leu Glu Asp Lys ThrGlu Arg Glu Leu Leu Glu Ser Tyr 20 25 30 atc gac ggg cgc att gtg gag ggctcg gat gca gag atc ggc atg tca 144 Ile Asp Gly Arg Ile Val Glu Gly SerAsp Ala Glu Ile Gly Met Ser 35 40 45 cct tgg cag gtg atg ctt ttc cgg aagagt ccc cag gag ctg ctg tgt 192 Pro Trp Gln Val Met Leu Phe Arg Lys SerPro Gln Glu Leu Leu Cys 50 55 60 ggg gcc agc ctc atc agt gac cgc tgg gtcctc acc gcc gcc cac tgc 240 Gly Ala Ser Leu Ile Ser Asp Arg Trp Val LeuThr Ala Ala His Cys 65 70 75 80 ctc ctg tac ccg ccc tgg gac aag aac ttcacc gag aat gac ctt ctg 288 Leu Leu Tyr Pro Pro Trp Asp Lys Asn Phe ThrGlu Asn Asp Leu Leu 85 90 95 gtg cgc att ggc aag cac tcc cgc acc agg tacgag cga aac att gaa 336 Val Arg Ile Gly Lys His Ser Arg Thr Arg Tyr GluArg Asn Ile Glu 100 105 110 aag ata tcc atg ttg gaa aag atc tac atc cacccc agg tac aac tgg 384 Lys Ile Ser Met Leu Glu Lys Ile Tyr Ile His ProArg Tyr Asn Trp 115 120 125 cgg gag aac ctg gac cgg gac att gcc ctg atgaag ctg aag aag cct 432 Arg Glu Asn Leu Asp Arg Asp Ile Ala Leu Met LysLeu Lys Lys Pro 130 135 140 gtt gcc ttc agt gac tac att cac cct gtg tgtctg ccc gac agg gag 480 Val Ala Phe Ser Asp Tyr Ile His Pro Val Cys LeuPro Asp Arg Glu 145 150 155 160 acg gca gcc agc ttg ctc cag gct gga tacaag ggg cgg gtg aca ggc 528 Thr Ala Ala Ser Leu Leu Gln Ala Gly Tyr LysGly Arg Val Thr Gly 165 170 175 tgg ggc aac ctg aag gag acg tgg aca gccaac gtt ggt aag ggg cag 576 Trp Gly Asn Leu Lys Glu Thr Trp Thr Ala AsnVal Gly Lys Gly Gln 180 185 190 ccc agt gtc ctg cag gtg gtg aac ctg cccatt gtg gag cgg ccg gtc 624 Pro Ser Val Leu Gln Val Val Asn Leu Pro IleVal Glu Arg Pro Val 195 200 205 tgc aag gac tcc acc cgg atc cgc atc actgac aac atg ttc tgt gct 672 Cys Lys Asp Ser Thr Arg Ile Arg Ile Thr AspAsn Met Phe Cys Ala 210 215 220 ggt tac aag cct gat gaa ggg aaa cga ggggat gcc tgt gaa ggt gac 720 Gly Tyr Lys Pro Asp Glu Gly Lys Arg Gly AspAla Cys Glu Gly Asp 225 230 235 240 agt ggg gga ccc ttt gtc atg aag agcccc ttt aac aac cgc tgg tat 768 Ser Gly Gly Pro Phe Val Met Lys Ser ProPhe Asn Asn Arg Trp Tyr 245 250 255 caa atg ggc atc gtc tca tgg ggt gaaggc tgt gac cgg gat ggg aaa 816 Gln Met Gly Ile Val Ser Trp Gly Glu GlyCys Asp Arg Asp Gly Lys 260 265 270 tat ggc ttc tac aca cat gtg ttc cgcctg aag aag tgg ata cag aag 864 Tyr Gly Phe Tyr Thr His Val Phe Arg LeuLys Lys Trp Ile Gln Lys 275 280 285 gtc att gat cag ttt gga gag 885 ValIle Asp Gln Phe Gly Glu 290 295 2 295 PRT Homo sapiens 2 Thr Phe Gly SerGly Glu Ala Asp Cys Gly Leu Arg Pro Leu Phe Glu 1 5 10 15 Lys Lys SerLeu Glu Asp Lys Thr Glu Arg Glu Leu Leu Glu Ser Tyr 20 25 30 Ile Asp GlyArg Ile Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser 35 40 45 Pro Trp GlnVal Met Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys 50 55 60 Gly Ala SerLeu Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys 65 70 75 80 Leu LeuTyr Pro Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu 85 90 95 Val ArgIle Gly Lys His Ser Arg Thr Arg Tyr Glu Arg Asn Ile Glu 100 105 110 LysIle Ser Met Leu Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp 115 120 125Arg Glu Asn Leu Asp Arg Asp Ile Ala Leu Met Lys Leu Lys Lys Pro 130 135140 Val Ala Phe Ser Asp Tyr Ile His Pro Val Cys Leu Pro Asp Arg Glu 145150 155 160 Thr Ala Ala Ser Leu Leu Gln Ala Gly Tyr Lys Gly Arg Val ThrGly 165 170 175 Trp Gly Asn Leu Lys Glu Thr Trp Thr Ala Asn Val Gly LysGly Gln 180 185 190 Pro Ser Val Leu Gln Val Val Asn Leu Pro Ile Val GluArg Pro Val 195 200 205 Cys Lys Asp Ser Thr Arg Ile Arg Ile Thr Asp AsnMet Phe Cys Ala 210 215 220 Gly Tyr Lys Pro Asp Glu Gly Lys Arg Gly AspAla Cys Glu Gly Asp 225 230 235 240 Ser Gly Gly Pro Phe Val Met Lys SerPro Phe Asn Asn Arg Trp Tyr 245 250 255 Gln Met Gly Ile Val Ser Trp GlyGlu Gly Cys Asp Arg Asp Gly Lys 260 265 270 Tyr Gly Phe Tyr Thr His ValPhe Arg Leu Lys Lys Trp Ile Gln Lys 275 280 285 Val Ile Asp Gln Phe GlyGlu 290 295

What is claimed is:
 1. A proteolytically active NP which has at least about 80% amino acid sequence homology with reference sequence thrombin and has a ratio of protein C activating activity to fibrinogen clotting activity that is less than about half of, or greater than about twice that of reference sequence thrombin, provided, however, that the novel polypeptide is not a naturally-occurring thrombin, thrombin K52E, thrombin R70E, thrombin R68E, thrombin K154A, thrombin K252E, thrombin K174E, thrombin R180E, thrombin D99A, thrombin D99N, thrombin E202Q, thrombin E25K, thrombin R245E, thrombin S205A, R197E, D199E, thrombin N151D, K154E, thrombin desP48P49W50, thrombin desE146T147W148, thrombin in which at least one amino acid residue within the thrombin activation site has been substituted or deleted, or thrombin in which loop F19-E25 is replaced by the equivalent loop from tissue plasminogen activator.
 2. An isolated proteolytically active NP which has at least about 80% amino acid sequence homology with reference sequence thrombin and has a ratio of protein C activating activity to fibrinogen clotting activity that is less than about half of, or greater than about twice that of reference sequence thrombin, provided, however, that the novel polypeptide is not thrombin K52E, thrombin R70E, thrombin R68E, thrombin K154A, thrombin K252E, thrombin K174E, thrombin R180E, thrombin D99A, thrombin D99N, thrombin E202Q, thrombin E25K, thrombin R245E, thrombin S205A, R197E, D199E, thrombin N151D, K154E, thrombin desP48P49QW50, thrombin desE146T147W148, thrombin in which at least one amino acid residue within the thrombin amino activation site has been substituted or deleted, or thrombin in which loop F19-E25 is replaced by the equivalent loop from tissue plasminogen activator.
 3. The NP of claim 1 wherein the NP is selected from a thrombin wherein one or more of residues W50, K52, D58, K65, H66, Y71, N74, K106, K107, S176, T177, W227, D193, K196, E202, E229, R233, D232, D234, K236, Y237 or F239 have been substituted, deleted or another residue inserted adjacent thereto.
 4. The NP of claim 3 wherein W50 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, E, Q, C, K, M, F, Y, P, R, and H.
 5. The NP of claim 3 wherein K52, K65, K106, K107 or K196 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, E, Q, C, M, F, Y, P, W, R, and H.
 6. The NP of claim 3 wherein D58, D193 or D234 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, N, E, Q, C, K, M, F, Y, P, W, R, and H.
 7. The NP of claim 3 wherein residues W50 and K52, E229 and W50, R233 and W50, R233 and K52, or R233 and E229 are substituted or deleted.
 8. The NP of claim 3 wherein H66 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, E, Q, C, K, M, F, Y, P, Wand R.
 9. The NP of claim 3 wherein Y71 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, E, Q, C, K, M, F, P, W, R, and H.
 10. The NP of claim 3 wherein N74 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, E, Q, C, K, M, F, Y, P, W, R, and H.
 11. The NP of claim 3 wherein E202 or E229 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, Q, C, K, M, F, Y, P, W, R, and H.
 12. The NP of claim 3 wherein the substitution, deletion or insertion is made only in the A or B chain.
 13. The NP of claim 3 wherein R233 is deleted or another residue selected from the following group has been substituted therefor or inserted immediately adjacent thereto: G, A, V, I, L, S, T, D, N, E, Q, C, K, M, F, Y, P, W, and H.
 14. The NP of claim 3 wherein residues D193, K196 and K52 are substituted or deleted.
 15. The NP of claim 3 wherein the residue is substituted and the substitution is with alanine.
 16. The NP of claim 3 wherein 2 or 3 of the residues are substituted.
 17. The NP of claim 3 which comprises B chain free of A chain.
 18. The NP of claim 3 which comprises A and B chain.
 19. The NP of claim 1 which possesses greater than about 2 times the residual proteolytic activity of reference thrombin when measured by hydrolysis of S-2238 in the presence of heparin-dependent AT-III inhibition.
 20. The NP of claim 3 wherein a heparin binding site residue is substituted.
 21. The NP of claim 20 wherein the residue is R89, R180, R245, K248 or K252.
 22. The NP of claim 3 which is K52A,R233A NP, E229D NP, E229F NP, E229S NP, E229W NP, E229Y NP, R233N NP, R233D NP, R233F NP, W50C NP, K52C NP, W50E NP or W50K NP.
 23. The NP of claim 1 wherein the thrombin has been substituted at residues K21, Q24, R70, R98 or K77, one or more of such residues have been deleted or another residue has been inserted immediately adjacent to one or more of such residues.
 24. The NP of claim 3 wherein the residue is E229 or R233.
 25. The NP of claim 1 wherein the residue is within about 10 Angstroms of the Cα of E229 or R233.
 26. A nucleic acid encoding the NP of claim
 1. 27. A replicable vector comprising the nucleic acid of claim
 26. 28. A recombinant cell comprising the vector of claim
 27. 29. A method comprising culturing the cell of claim 28 and recovering the NP from the cell culture.
 30. The method of claim 29 wherein the NP is expressed in the cell culture as a soluble polypeptide.
 31. The method of claim 29 wherein the NP is expressed in the cell culture as the B chain alone.
 32. The method of claim 29 wherein the nucleic acid encodes A and B sequences each one of which is independently ligated to nucleic acid encoding a signal sequence and the nucleic acid is coexpressed in the same host cell.
 33. A method for the treatment of thrombotic diseases or conditions comprising administering a therapeutically effective amount of a proteolytically active NP which is at least about 80% homologous by amino acid sequence with reference sequence thrombin and has a ratio of protein C activating activity to fibrinogen clotting activity that is greater than about twice that of reference sequence thrombin.
 34. The method of claim 33 wherein the NP possesses greater than about twice the residual proteolytic activity of reference thrombin when measured by hydrolysis of S-2238 in the presence of heparin-dependent AT-III inhibition.
 35. The method of claim 33 wherein the thrombotic disease or condition is cardiac bypass surgery.
 36. The method of claim 33 wherein the NP is devoid of detectable fibrinogen clotting activity.
 37. The method of claim 36 wherein the NP retains at least about 5% of wild-type thrombin protein C activating activity.
 38. The method of claim 37 wherein the NP is K52A, R233A NP, E229D NP, E229F NP, E229S NP, E229W NP, E229Y NP, R233D NP, R233N NP, R233F NP, W50C NP, K52C NP, W50E NP, or W50K NP.
 39. An NP which is at least about 80% homologous by amino acid sequence with reference sequence thrombin in which at least one amino acid residue has been substituted, deleted or inserted immediately adjacent to thrombin residues R20, K21, S22 Q24, E25, D35, W250, D51, K52, N53, F54, T55, N57, D58, K65, H66, R68, T69, R70, Y71, R73, N74, K77, E82, E83, R89, R93, R94, R98, K106, K107, D113, C119, D122, R123, E124, S128, Q131, K145, E146, T147, W148, T149, N151, K154, S158, E169, K174, D175, S176, T177, R178, I179, R180, D183, D193, K196, D199, A200, C201, E202, N216, N217, W227, G228, E229, G230, C231, D232, R233, D234, G235, K236, Y237, G238, F239, R245, K248, Q251, R245, K248, W249, Q251, K252, D255, or Q256, provided, however, that the NP is not thrombin K52E, thrombin R70E, thrombin R68E, thrombin K154A, thrombin K252E, thrombin K174E, thrombin R180E, thrombin D99A, thrombin D99N, thrombin E202Q, thrombin E25K, thrombin R245E, thrombin S205A, R197E, D199E, thrombin N151D, K154E, thrombin desP48P49QW50, thrombin desE146T147W148, thrombin in which at least one amino acid residue within the thrombin amino activation site has been inserted, substituted or deleted, or thrombin in which loop F19-E25 is replaced by the equivalent loop from tissue plasminogen activator. 